Non-shared binding shielding polyma

Abstract

The present invention relates to novel anionic polymers that are useful as shields for positively charged protein or polycation-based non-viral vectors for the delivery of active ingredients, including proteins or nucleic acids, to cells.

Description

[Technical field]

[0001] This application claims the benefit of European patent application EP21382665.4, filed on July 22, 2021.

[0002] The present disclosure relates to novel anionic polymers that are useful as shields for positively charged protein or polycation-based non-viral vectors for delivering active ingredients, including proteins or nucleic acids, to cells. [Background technology]

[0003] Delivery of active ingredients to target sites in cells requires a suitable delivery carrier that can provide adequate protection and efficiently deliver active ingredients to specific tissues in the body.Delivery carriers must nevertheless overcome various extracellular and intracellular barriers to reach their target sites in cells.Although viral vectors are more efficient for gene delivery than non-viral vectors, their use entails several risks, including toxicity, immunogenicity, and size limitations of genetic cargo.In general, non-viral vectors are safer and easier for large-scale production, although their transfection efficiency is relatively low.

[0004] The use of cationic polymers as non-viral synthetic carriers for delivering active ingredients, especially nucleic acids, to target cells has attracted considerable attention.

[0005] Cationic polymers such as polyethyleneimine (PEI), poly(β-amino ester), polyamidoamine, chitosan, and other polyamines such as PAsp(DET) can form nanoparticles (polycation-based non-viral vectors) that carry and protect active ingredients, especially nucleic acids. Thus, these cationic polymers can carry and protect the active ingredient as it enters the cell membrane and finally reaches the nucleus.

[0006] Nevertheless, one of the main reasons why the development of polycation-based non-viral vectors is limited is that cationic polymers are required to exhibit different functions at different stages of the delivery process. For example, the vector may need to have high amine density to overcome the endosomal membrane barrier, since the protonated potential of nanoparticles may cause endosomal destabilization and allow release in the cytoplasmic medium. Conversely, the positively charged properties of nanoparticles or proteins may cause aggregation in the bloodstream and non-specific interactions with negatively charged serum components, thereby causing thrombosis in capillaries or preventing proteins from performing their physiological functions. This highly positively charged particle (polyplex or protein) may also have the risk of inducing high cytotoxicity and excessive immune response. Moreover, these positively charged nanoparticles may cause severe serum inhibition and are rapidly cleared from the blood, which hinders their application in vivo.

[0007] A well-known attempt to solve these problems is the shielding of the positive charges by covering the surface of the nanoparticles with polyethylene glycol (PEG). However, the presence of covalently attached PEG significantly reduces the transfection efficiency in the case of polyplexes, because the neutral surface of the nanoparticles can reduce the cellular uptake efficiency, or in the case of proteins, because the active site space is hindered, which can cause activity loss. It is also well known that PEG generates anti-PEG antibodies, which can cause immunogenic and allergic reactions.

[0008] Therefore, from what is known in the art, there remains a need to find new shielding strategies that overcome the above problems. Summary of the Invention

[0009] Therefore, the inventors have designed novel anionic polymers useful as shields for positively charged protein or polycation based non-viral vectors for delivery of active ingredients including proteins or nucleic acids to cells with low cytotoxicity, high efficiency, suitable plasma half-life, enhanced permeability and retention, high solubility in aqueous solutions, high stability since aggregation problems in bloodstream, potential different cell and tissue targeting are limited or completely suppressed.

[0010] In the context of the present invention, the term "anionically charged polymer", "polyanionically charged polymer", "anionic polymer" or equivalents refer to a polymer that contains natural or non-natural anionic amino acids that contain a net negative charge at basic or physiological pH, and may also contain, for example, glutamic acid, aspartic acid, or combinations thereof, i.e., anionic groups that are already anionic upon loss of a hydrogen ion, but would become neutral upon gaining a hydrogen ion. Polypeptides with anionic groups in the side chain also include polypeptide derivatives obtained by peptide bonding of known amino acids with acidic side chains (e.g., glutamic acid, aspartic acid), as well as polypeptides obtained by peptide bonding of any amino acid and subsequent substitution in the side chain, resulting in an anionic charged group.

[0011] As used herein, the term "non-covalent bonds" refers to bonds that do not involve the sharing of electrons, but rather a more diffuse variation of electromagnetic interactions between molecules. Non-covalent bonds can be classified into various categories, such as electrostatic interactions, π interactions, van der Waals forces, hydrogen bonds, and hydrophobic effects.

[0012] Thus, a first aspect of the present invention relates to an anionic polymer comprising the following formula (Ia) or (Ib), a pharma- ceutically acceptable salt thereof, or any stereoisomer or mixture of stereoisomers of any of the compounds of formula (Ia) or formula (Ib), including homopolypeptides or random or block or graft copolypeptides, or a pharma- ceutically acceptable salt thereof: [ka] wherein Y is selected from the group consisting of -CO(CH2)p-CO- and -CO-(CH2)qSS-(CH2)r-CO-; Z is selected from the group consisting of a single bond, -CO-(CH2)qSS-(CH2)r-NH-(R5)z-, -CO(CH2)pNH-(R5)z-, -CO-(CH2)p-CO-NH-(CH2)qSS-(CH2)r-NH-(R5)z-, and -(R5)z-; ○ is an integer selected from 1 to 2; p, q, and r are each independently an integer selected from 1 to 6; R5 is a random or block copolymer comprising at least two different repeat units selected from the group consisting of (II), (III), (IV), (V), (VI), (VII), (VIII), and (IX): [ka] a, b, c, d, e, f, g, and h are each independently an integer selected from 0 to 20; provided that at least two of a, b, c, d, e, f, g, and h are different from 0; z is an integer selected from 5 to 100; X is selected from N, S, and O; R1 is selected from the group consisting of H and (C1-C6)alkyl, with the proviso that R1 is absent when X is O; R3 and R4 are each independently selected from the group consisting of H and -CH3; m is an integer selected from 5 to 250; n is an integer selected from 3 to 200; However, the m:n ratio is in the range of 1:8 to 30:1; R2 is selected from H and a radical selected from the group consisting of (X), (XI), (XII), and (XIII); [ka] In the formula, s, t, u, and v are each independently an integer selected from 1 to 4; wherein " / " indicates that the sequential order of the monomeric repeat units defined by the brackets on either side of the symbol is arbitrary, and thus, although the repeat units defined by the brackets in formulas Ia and Ib are shown in a particular order for convenience of illustration, the repeat units may be present in any order, and the repeat units may be present in blocks or randomly; wherein in formula Ib, the sequential order of the repeat units of each of formulae (II), (III), (IV), (V), (VI), (VII), (VIII), and (IX) in R5 and the repeat units indicated in square brackets with integer n may be present in blocks or randomly; A and A' are each H, OH, linear or branched chain -(C1-C6) alkyl, linear or branched chain -CO(C1-C6) alkyl, -(C5-C 10 )Aryl, -(C5-C 10 )Heteroaryl, -(C6-C 10 ) aralkyl, -(C1-C6) alkyl-O-(C5-C 10 )Aryl, -(C5-C 10 )heterocycloalkyl, -(C1-C 10 )Alkoxy, -(C6-C 10 )Aryloxy, -(C6-C 10 )Aralkoxy, -(C5~C 10 )Heteroaralkoxy, -(C1-C 10 )Alkyl-O-(C6-C 10) independently selected from aryloxy, an amine protecting group, a natural or unnatural alpha amino acid, a carboxyl protecting group, a labeling or imaging agent, and a cell targeting agent; each of A and A' is independently optionally substituted with one or more groups selected from the group consisting of -OH, halogen, -CF3, -NH2, -NH-(C1-C4)alkyl, NR6R7, -NH-CO-(C1-C6)alkyl, -(C1-C6)alkyl, -NO2, -N3, -CO-(C1-C6)alkyl, -CO-O-(C1-C6)alkyl, -SO3H, -SON2NH2, -SO2-N((C1-C6)alkyl)2, -COOH, CONH2, and -CON((C1-C6)alkyl)2; R6 and R7 are independently selected from the group consisting of H, -(C1-C4)alkyl, and an amine protecting group.

[0013] According to a second aspect of the present invention, a) a cationic polymer covalently or electrostatically associated with at least one active agent, preferably selected from the group consisting of pharmaceutical active agents, veterinary active agents, cosmetic active agents, diagnostic active agents, nucleic acids, peptides, antibodies, aptamers, proteins, and mixtures thereof, wherein the cationic polymer and the at least one active agent form positively charged nanoparticles; b) at least one anionic polymer of formula (Ia) or (Ib) as defined herein, which interacts electrostatically with the nanoparticles; A polymer complex is provided comprising:

[0014] According to another aspect of the present invention, a) a positively charged protein; b) at least one anionic polymer of formula (Ia) or (Ib) as defined herein, which interacts electrostatically with the protein; or instead a') anionically charged proteins that are covalently or electrostatically bound to cationic polymers that form positively charged nanoparticles; b') at least one anionic polymer of formula (Ia) or (Ib) according to any one of claims 1 to 7, which interacts electrostatically with the positively charged nanoparticles of a'), A protein-based complex comprising:

[0015] According to a fourth aspect of the present invention there is provided a composition comprising at least one polymer complex or protein-based complex as defined herein and one or more suitable (e.g. pharma- ceutical , diagnostic, veterinary or cosmetic) acceptable excipients or carriers.

[0016] In a further aspect, the present invention relates to a polymer complex, a protein-based complex, or a pharmaceutical composition as defined herein for use as a medicament.

[0017] Alternatively, this embodiment may be formulated as a therapeutic product, being a polymer complex as defined herein, or a protein-based complex as defined herein, or a composition as defined herein, for use in medicine.

[0018] A further aspect of the present invention is (i) for use as a transfection reagent for transfecting at least one active agent into a cell; or (ii) for use in the in vivo or ex vivo production of biological products encoding recombinant proteins, peptides, or antibodies, or in the production of recombinant viruses; (iii) for use as a therapeutic or prophylactic vaccine against viral infection or as a therapeutic vaccine against cancer; or (iv) for use in genome engineering, cell reprogramming, cell differentiation, or gene editing; The present invention relates to a polymer complex as defined herein or to a pharmaceutical composition comprising same.

[0019] A further aspect of the present invention relates to the protein-based complexes as defined herein or pharmaceutical compositions comprising them for use in protein-based therapy, in particular for use as a therapeutic or prophylactic protein-based vaccine against viral infections or as a therapeutic protein-based vaccine against cancer.

[0020] The invention also provides a device that may be suitable for delivering, for example, an active agent, preferably a nucleic acid or a protein, into a cell, tissue or extracellular space, the device comprising a polymer complex as defined herein or a composition comprising same. This aspect can also be formulated as a device for use in a method of delivering an active agent, for example a nucleic acid or a protein, into a cell, tissue or extracellular space, the device comprising a polymer complex as defined herein, a protein-based complex as defined herein, or a composition comprising same as defined herein.

[0021] As will be appreciated by those skilled in the art, the appropriate device for delivering active agent into cells will depend on the formulation of the selected composition or pharmaceutical composition and / or the desired administration site.For example, if the composition formulation is suitable for injection into a subject, the device can be a syringe.As another example, if the desired administration site is a cell culture medium, the device can be a sterile pipette.As yet another example, if the desired administration site is a vein or artery, the device can be a graft.As yet another example, if the desired administration site is a subcutaneous or organ-specific depot, the device can be a surgical implant.

[0022] The delivery device of the present invention may be utilized in therapy (gene therapy) in which a nucleic acid of interest is introduced into cells responsible for any of a variety of diseases.

[0023] According to a further aspect of the present invention, a method for delivering protein or nucleic acid into target cells, tissues or extracellular space is provided, the method comprising administering a solution containing a polymer complex, a protein-based complex or a composition as defined herein to an animal, including a human, whereby the complex can be introduced into target cells, tissues or extracellular space. In the case of a polymer complex or a protein-based complex carrying an active ingredient that has its action in the intracellular space, the transport is carried out via cellular internalization, which transfers the complex to the cytoplasm by an internalization mechanism; dissociates the complex inside the cell; and releases the protein or active ingredient in the cytoplasm. In the case of a protein complex that has its action in the extracellular space, internalization is not aimed for or required.

[0024] This aspect can be formulated as the use of a polymer complex, a protein-based complex or a pharmaceutical composition disclosed herein in a method for delivery of a nucleic acid into a target cell, the method comprising contacting a solution containing the polymer complex, the protein-based complex or the composition defined herein with the target cell, tissue or extracellular space such that the complex can be introduced into the target cell, tissue or extracellular space; translocating the complex from the endosome to the cytoplasm; dissociating the complex in the cell; and releasing the nucleic acid in the cytoplasm.

[0025] Alternatively, this embodiment can be formulated as a polymer complex as defined herein, a protein-based complex as defined herein, or a composition as defined herein for use in a method of delivering a nucleic acid or protein into a target cell, tissue, or extracellular space, the method comprising contacting a target cell with a solution comprising a polymer complex as defined herein, a protein-based complex as defined herein, or a composition as defined herein, such that the complex may be introduced into the target cell, tissue, or extracellular space; translocating the complex from an endosome to the cytoplasm; dissociating the complex in the cell; and releasing the nucleic acid or protein into the cytoplasm.

[0026] A further aspect relates to a method of transfecting a cell comprising contacting the cell with a polymeric complex as defined herein, a protein-based complex as defined herein, or a composition as defined herein.

[0027] In another aspect, the present disclosure relates to a process for the synthesis of the compound of formula (Ia) or (Ib) of the first aspect of the present disclosure or any embodiment thereof, which generally comprises polymerizing N-carboxyanhydrides (NCAs) of amino acids, protected or unprotected, known per se, to produce poly(amino acids), or protected polyamino acid esters, carbamates, S-alkylsulfonyl, or trifluoroacetyl derivatives. A deprotection step or thiol exchange must then be carried out by methods well known to those skilled in the art. The different radicals present in the repeating units can be introduced in the desired ratio by changing the ratio of each block or random copolymer.

[0028] According to this aspect, there is provided a process for the synthesis of a compound of formula (I) of the first aspect of the disclosure or any embodiment thereof, the process comprising: i) reacting an initiator in the form of an amine or an ammonium salt of tetrafluoroboric acid or trifluoroacetate; i.1) using a suitable N-carboxyanhydride (NCA); alternatively, reacting the initiator in the form of an amine or tetrafluoroboric acid or trifluoroacetate ammonium salt of step i) successively with a suitable N-carboxyanhydride to obtain a block copolymer; i.2) Alternatively, reacting the initiator in the form of an amine or tetrafluoroboric acid or trifluoroacetate ammonium salt of step i) with a suitable NCA mixture in a statistical manner to obtain a random copolymer; ii) optionally reacting the amine group at the N-terminal position with an amine-reactive group to introduce R1; iii) optionally, orthogonally removing amino acid side chain protecting groups; iv) optionally reacting an amine, thiol, disulfide, S-alkylsulfonyl group or carboxylic acid group at a side chain terminal position with an amine or carboxylic acid reactive group to introduce structural extension, conjugation, labeling or shielding at R2; v) purifying the product obtained in step i), ii) or iii) optionally by fractionation, precipitation, ultrafiltration, dialysis, size exclusion chromatography, affinity chromatography or tangential flow filtration; Includes.

[0029] Said step i) may comprise: a) ring-opening polymerization of amino acid N-carboxyanilide (NCA) monomers by reacting an initiator in the form of an amine or a tetrafluoroborate or an ammonium trifluoroacetate salt with the selected NCA, where the monomer / initiator ratio allows for control of the degree of polymerization (DP); b) step-growth polymerization, where block copolypeptides are prepared after the polymerization reaction a') in a sequential manner, allowing the initial NCA monomers to be consumed, and the resulting product may or may not be purified before adding the next monomer to build the next polypeptide block; or c) statistical polymerization of a'), where random copolypeptides are prepared after the polymerization reaction in a statistical manner by mixing all NCA monomers before starting the polymerization by adding an initiator in the form of an amine or a tetrafluoroborate or an ammonium trifluoroacetate salt.

[0030] Step ii) above corresponds to end-capping, where an amine group at the N-terminal position reacts with an amine-reactive group to introduce R1.

[0031] Step iii) above corresponds to an exchange reaction or deprotection, in which the amino acid side chain is orthogonally removed, depending on the protecting group.

[0032] Step iv) corresponds to conjugation, whereby an amine or carboxylic acid group is reacted at the side chain terminal position by chloroacetylation, methylation, thiol exchange, nucleophilic substitution, or peptide coupling reactions, optionally in a sequential manner, to achieve a masked moiety.

[0033] According to another aspect of the present invention there is provided a process for preparing a compound structurally different to a compound of formula I as defined herein, the process comprising the steps of: i. using a compound of formula I as defined herein as a starting compound; ii. subjecting the compound of step (i) to a structural modification to obtain a compound structurally different from the compound of formula I; Includes.

[0034] In a further aspect of the invention there is provided the use of a compound of formula I as defined herein for the preparation of a compound which is structurally different to a compound of formula I.

[0035] Non-limiting examples of the present disclosure are described below with reference to the accompanying drawings: [Brief description of the drawings]

[0036] [Figure 1]The results of polyplexes PXN1_8_0.5mRNA (top left), PXN1_8_0.5pDNA (top right) with shielding polymer V1 analyzed by the agarose gel electrophoresis technique are shown. This technique shows in a qualitative way the ability of polyplexes to complex with genetic material (RNA or DNA). It also shows the ability to release genetic material at low and high concentrations and in the presence of low and high concentrations of a polyanionic competitor (heparin). The lane labeled M is seeded with free mRNA and lane 4 is seeded with pDNA. As can be seen, the free genetic material glows under a UV transilluminator. In lanes 1 and 5, polyplexes are seeded and it can be seen that when polycations are present and polyplexes are formed, the genetic material is captured and no signal can be observed. In lanes 2 and 6, polyplexes are seeded in the presence of low concentrations of heparin competitor and show no release in these conditions. In lanes 3 and 7, polyplexes are seeded with high concentrations of anionic heparin competitors, in which case release of genetic material is observed. This behavior is ideal because polyplexes need to be stable at low concentrations of the competitor molecule outside the cell, yet unstable enough to release the cargo when an intracellular stimulus is applied. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] All terms used herein in this application are to be understood in their ordinary meaning as known in the art unless otherwise specified. Other, more specific definitions of certain terms used in this application are set forth below and are intended to be applied uniformly throughout the specification and claims, unless a definition expressly set forth otherwise provides a broader definition.

[0038] As used herein, the indefinite articles "a" and "an" are equivalent to "at least one" or "one or more." Unless otherwise specified, definite articles such as "the" as used herein also include the plural form of a noun.

[0039] The term "halogen" or "halo" as used herein means fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine, more preferably fluorine and chlorine.

[0040] The term "alkenyl" refers to an organic group composed of carbon and hydrogen atoms, including at least one double covalent bond between two carbons. Typically, "alkenyl" as used in this disclosure refers to an organic group containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 carbon atoms, or any range between or including any two of the aforementioned values. In some cases, the alkenyl group is conjugated, in other cases, the alkenyl group is not conjugated, and in yet other cases, the alkenyl group may have conjugated and non-conjugated extensions. Furthermore, when there are more than two carbons, the carbons may be connected in a linear fashion, or when there are more than three carbons, the carbons may be linked in a branched fashion, such that the parent chain includes one or more secondary, tertiary, or quaternary carbons. The alkenyl may be substituted or unsubstituted.

[0041] The term "alkyl" as used herein refers to a saturated, straight or branched hydrocarbon chain, i.e., it refers to an organic group composed of carbon and hydrogen atoms, containing one covalent bond between the carbons. Typically, "alkyl" as used in this disclosure refers to an organic group containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 carbon atoms, or any range between or including any two of the aforementioned values. Examples of alkyl groups with 1 to 12 carbon atoms may include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, n-hexyl, decyl, and undecyl groups.

[0042] If there is more than one carbon, the carbons may be connected in a linear fashion, and if there are more than two carbons, the carbons may be linked in a branched fashion, such that the parent chain contains one or more secondary, tertiary, or quaternary carbons. Alkyl may be substituted or unsubstituted.

[0043] The term "alkynyl" refers to an organic group composed of carbon and hydrogen atoms, including a triple covalent bond between two carbons. Typically, "alkynyl" as used in this disclosure refers to an organic group containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 carbon atoms, or any range between or including any two of the aforementioned values. C2-alkynyl can form a triple bond to a carbon of the parent chain, while alkynyl groups of three or more carbons can contain multiple triple bonds. If there are more than three carbons, the carbons can be connected in a linear fashion, and if there are more than four carbons, the carbons can be linked in a branched fashion, such that the parent chain includes one or more secondary, tertiary, or quaternary carbons. Alkynyl can be substituted or unsubstituted.

[0044] Terms commonly represented by the notation "Cx-Cy" (where x and y are integers and y>x) before a functional group, such as "C1-C12 alkyl", refer to a range of numbers of carbon atoms. For purposes of this disclosure, the range designated by "Cx-Cy" (where x and y are integers and y>x) is not limited to the range designated, but includes the range designated by "Cx-Cy" and all possible ranges that fall within that range (where x and y are integers and y>x). For example, the term "C1-C4" provides explicit support of a range of 1-4 carbon atoms, but also provides implicit support of the range encompassed by 1-4 carbon atoms, such as 1-2 carbon atoms, 1-3 carbon atoms, 2-3 carbon atoms, 2-4 carbon atoms, and 3-4.

[0045] The term "fluoroalkyl," as used herein, refers to an alkyl group, as defined herein, that is substituted one or more times with one or more fluorohalos, preferably perfluorinated.

[0046] The term "alkoxy" as used herein refers to the group "alkyl-O-", where alkyl is defined above.

[0047] The term "substituted" means that one or more hydrogen atoms on a specified atom or group are replaced with one selected from a specified group, provided that the normal valence of the specified atom under the present circumstances is not exceeded. Combinations of substituents and / or variables are permissible.

[0048] The term "optionally substituted" means that the number of substituents can be equal to or different from zero.Unless otherwise indicated, an optionally substituted group can be substituted with as many optional substituents as it can accommodate by replacing hydrogen atoms with non-hydrogen substituents on any available carbon or nitrogen atom.Groups in the compounds according to the present invention can be substituted with 1, 2, 3, 4 or 5 identical or different substituents, particularly 1, 2 or 3 substituents.

[0049] In those embodiments of the invention where the substitution or unsubstitution of a particular group is not specified, i.e., where no particular substitution for the group is indicated, nor is the group indicated as being unsubstituted, it is to be understood that the possible substitution of this group is in the broadest scope as defined herein.

[0050] The term "disorder" as used herein is intended to be generally synonymous with, and is used interchangeably with, the terms "disease," "syndrome," and "condition" (as in medical condition), in that all reflect an abnormal condition of the human or animal body or one of its parts that impairs normal function and typically exhibit characteristic signs and symptoms.

[0051] As used herein, the terms "pharmaceutical acceptable carrier", "pharmaceutical acceptable excipient", "physiologically acceptable carrier", or "physiologically acceptable excipient" refer to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulant. Each component must be "pharmaceutical acceptable" in the sense of being compatible with the other components of a pharmaceutical formulation. It must also be suitable for use in contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications, consistent with a reasonable benefit / risk ratio.

[0052] The terms "cosmetically acceptable carrier" or "dermatologically acceptable carrier", as used interchangeably herein, refer to excipients or carriers that are suitable for use in contact with human skin without undue toxicity, incompatibility, instability, or allergic response.

[0053] The term "therapeutically acceptable" refers to compounds that are suitable for use in contact with the tissues of a patient without undue toxicity, irritation, allergic response, or immunogenicity, and that are effective for their intended use, consistent with a reasonable benefit / risk ratio.

[0054] The term "pharmaceutically, cosmetically, or diagnostically acceptable salts" includes commonly used non-toxic salts. The preparation of pharmaceutically, cosmetically, or diagnostically acceptable salts of the compounds of the present invention can be carried out by methods well known in the art. In general, such salts can be prepared by reacting the free acid or base form of the compounds of the present invention with a stoichiometric amount of a suitable base or acid, respectively, in a suitable solvent such as water, an organic solvent, or a mixture thereof.

[0055] Examples of pharma- ceutically , cosmetically , or diagnostically acceptable salts include those of inorganic acids, such as hydrochloric, hydrobromic, sulfuric, nitric, hydroiodic, metaphosphoric, or phosphoric acids, as well as organic acids, such as succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, trifluoroacetic, malic, lactic, formic, propionic, glycolic, gluconic, camphorsulfuric, isothionic, mucic, gentisic, isonicotinic, saccharic, glucuronic, furoic, glutamic, ascorbic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), ethylenediamine, ethylenediamine, ethylenediaminetetraacetic ... These include acid addition salts formed with benzenesulfonic acid, pantothenic acid, stearic acid, sulfinilic acid, alginic acid and galacturonic acid; and arylsulfonic acids, such as benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, methanesulfonic acid or naphthalenesulfonic acid; base addition salts formed with alkali metals and alkaline earth metals, such as N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), lysine and procaine, and salts formed internally. The compounds of the present invention and their salts may differ in some physical properties, but for the purposes of the present invention they are equivalent.

[0056] As used herein, the term "pharmacologically active agent" refers to an agent that has pharmacological activity and is used to cure, alleviate, treat or prevent disease in mammals, particularly humans. The term "cosmetically active agent" refers to an agent that does not provide any medical treatment but is used for cosmetic purposes, such as improving the appearance, preserving, conditioning, cleaning, coloring or protecting the skin, nails or hair.

[0057] The term "diagnostic composition" refers to a composition suitable for use in diagnosis, particularly imaging diagnostic techniques. The term "diagnostically effective amount" as used herein refers to an effective amount of the detection polymer that, when administered, is sufficient for the diagnosis of a disease or disorder, particularly for imaging diagnostics as a contrast imaging agent. The dose of the detection polymer administered will, of course, be determined by the particular circumstances surrounding the case, including the polymer administered, the route of administration, the particular condition being diagnosed, and similar considerations. The diagnostic composition of the present invention includes one or more diagnostically acceptable excipients or carriers. The term "diagnostically acceptable" refers to an excipient or carrier suitable for use in diagnostic techniques to prepare a composition having diagnostic use, particularly by imaging diagnostic use. Detection of these diagnostic agents in the patient's body can be performed by well-known techniques used in imaging diagnostics, such as magnetic resonance imaging (MRI) and X-ray imaging diagnostics.

[0058] As used herein, the phrase "natural amino acid" refers to any of the 20 amino acids that naturally occur in proteins. Such natural amino acids include the non-polar or hydrophobic amino acids glycine, alanine, valine, leucine isoleucine, methionine, phenylalanine, tryptophan, and proline. Cysteine ​​may be classified as non-polar or hydrophobic, or as polar. Natural amino acids also include polar or hydrophilic amino acids such as tyrosine, serine, threonine, aspartic acid (also known as aspartate when charged), glutamic acid (also known as glutamate when charged), asparagine, and glutamine. Certain polar or hydrophilic amino acids have a charged side chain, depending on the pH of the environment. Such charged amino acids include lysine, arginine, and histidine. Those skilled in the art will recognize that protection of polar or hydrophilic amino acid side chains can make the amino acid non-polar. For example, suitably protected tyrosine hydroxyl groups can render the tyrosine non-polar and hydrophobic by protecting the hydroxyl group.

[0059] As used herein, the phrase "unnatural amino acid" refers to an amino acid that is not included in the list of the 20 amino acids that naturally occur in proteins, as described above. Such amino acids include the D-isomers of any of the 19 naturally occurring amino acids, and glycine is non-chiral. Unnatural amino acids also include homoserine and ornithine. Other unnatural amino acid side chains are well known to those skilled in the art, including unnatural aliphatic side chains. Other unnatural amino acids include modified amino acids, including N-alkylated, cyclized, phosphorylated, acetylated, amidated, azidylated, labeled, etc.

[0060] As used herein, the terms "treat," "treating," and "treatment" refer to ameliorating the symptoms associated with a disease or disorder, including preventing or delaying the onset of a symptom of a disease or disorder and / or reducing the severity or frequency of a symptom of a disease or disorder.

[0061] As used herein, the term "peptide" refers to a molecule that contains two or more consecutive amino acids linked together via peptide bonds. The term peptide includes oligopeptides and polypeptides. The term "protein" refers to large peptides, particularly peptides having at least about 50 amino acids. For purposes of the present invention, the terms peptide and protein are used interchangeably.

[0062] As used herein, the term "repeat unit" or "block" refers to a repeating monomer unit. A repeat unit or block may be composed of one monomer, or may be composed of one or more monomers randomly or in blocks, resulting in a "mixed block". Thus, formulas Ia and Ib defined above encompass compounds that may contain repeat units defined by square brackets, each of which may contain the same or different substituents. When the monomer units present in the same repeat unit are the same, the repeat unit is a "homopolymer", and when the monomer units present in the same repeat unit contain different substituents, the repeat unit is a "copolymer", which may be a "random copolymer" or a "block copolymer".

[0063] For purposes of the present invention, the term "homopolymer" refers to a polymer derived from a single monomer. The term "copolymer" as used herein refers to a polymer derived from two or more monomers. A copolymer may be a random or block copolymer. The term "random copolymer" as used herein refers to a copolymer in which the monomer units are randomly arranged in the polymer molecule. The term "block copolymer" as used herein refers to a copolymer that includes at least two different monomer units that upon polymerization form at least two chemically distinct regions, segments, or blocks that are chemically distinguishable from one another. The term block copolymer includes linear block copolymers, multiblock copolymers, and star block copolymers.

[0064] One of ordinary skill in the art will recognize that repeat units are defined by brackets ("[]") drawn around the repeating monomeric unit. The number (or letter representing a numerical range) to the right of the brackets represents the number of monomeric units present in the polymer chain.

[0065] Using appropriate surface functional groups, the compounds of the present disclosure may be further modified with cell targeting groups and / or penetration enhancers that can actively target cells and aid in cell entry, resulting in conjugates with improved cell-specific delivery.

[0066] As used herein, the term "protecting group" refers to a group of atoms that, when attached to a reactive group in a molecule, masks, reduces or prevents the reactivity of the group.Protective groups for carboxyl and amino groups are described, for example, in TW Green and PG M Huts, Protective Groups in Organic Chemistry (Wiley, 3rd ed. 1999), Chapter 5 (pp. 369-451) and Chapter 7 (pp. 495-653), respectively.Any suitable amine protecting group known in the art may be used without limitation, examples of which include acyl-based groups, carbamate-based groups, imide-based groups, sulfonamide-based groups, etc. Among these, methyloxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, t-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (FMOC), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl group (Troc), benzoyl (Bz), benzyl (Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trimethylsilylethyloxycarbonyl (Teoc), benzhydryl, triphenylmethyl (trityl), (4-methoxyphenyl)diphenylmethyl (MMT), dimethoxytrityl (DMT), and diphenylphosphino group are preferred.

[0067] Introduction and removal of amino protecting groups can be carried out by standard methods, such as those described in TW Green and PGM Huts, Protective Groups in Organic Chemistry, Wiley, 3rd ed. 1999, Chapter 7 (pp. 495-653).

[0068] Any suitable carboxy protecting group known in the art may be used without limitation. Representative carboxy protecting groups include alkyl, aryl or benzyl esters, silyl esters, amides or hydrazides. In certain embodiments, the carboxy protecting group is selected from the group consisting of -(C1-C6) alkyl, benzyl, p-methoxyphenyl, trimethylsilyl and [2-(trimethylsilyl)ethoxy]methyl (SEM).

[0069] The introduction and removal of these protecting groups can be carried out by standard methods, such as those described in TW Green and PGM Huts, Protective Groups in Organic Chemistry, Wiley, 3rd ed. 1999, Chapter 5 (pp. 369-451).

[0070] As used herein, the term "initiator" refers to a chemical molecule used for initiation of the ring-opening polymerization (ROP) reaction of α-amino acid N-carboxyanhydrides via a normal amine mechanism, where the initiator is incorporated into the backbone of the resulting polyamino acid. The initiator may contain one or more nucleophilic groups capable of initiating the ROP reaction, and thus the initiator may be mono- or polyfunctional, respectively, resulting in one or several terminal X groups in the polymer of the invention, respectively.

[0071] The term "moiety" refers to a specific segment or functional group of a molecule or compound.

[0072] As used herein, the term "subject" refers to any mammal, including both humans and other mammals.

[0073] The term "nanoparticle" as used herein refers to a particle having at least two dimensions at the nanoscale, particularly all three dimensions at the nanoscale. In particular, when the nanoparticle is substantially rod-shaped with a substantially circular cross section, such as a nanowire or nanotube, "nanoparticle" refers to a particle having at least two dimensions at the nanoscale, the two dimensions being the cross section of the nanoparticle.

[0074] As used herein, the term "size" refers to a characteristic physical dimension. For example, for a substantially spherical nanoparticle, the size of the nanoparticle corresponds to the diameter of the nanoparticle. For a substantially rod-like nanoparticle with a substantially circular cross section, such as a nanowire or nanotube, the size of the nanoparticle corresponds to the diameter of the cross section of the nanoparticle. For a substantially box-shaped nanoparticle, such as a nanocube, nanobox, or nanocage, the size of the nanoparticle corresponds to the maximum edge length. When referring to a set of nanoparticles as being of a particular size, it is contemplated that the set of nanoparticles may have a distribution of sizes around the specified size. Thus, as used herein, the size of a set of nanoparticles may refer to the mode of the size distribution, such as the peak size of the size distribution.

[0075] The term "polydispersity index" (PDI) is used as a measure of the broadness of the molecular weight distribution. The higher the PDI, the broader the molecular weight. The PDI of a polymer is calculated as the ratio of the weight average (MW) to the number average (Mn) molecular weight.

number

[0076] The compounds of formula (Ia) and (Ib) may exist as geometric isomers (i.e., cis-trans isomers), stereoisomers such as optical isomers or diastereomers, and tautomers. It should therefore be understood that the definition of the compounds of formula (Ia) and (Ib) includes all individual isomers corresponding to structural formula (Ia) and (Ib), including cis-trans isomers, stereoisomers and tautomers, as well as racemic mixtures thereof, and pharma- ceutically acceptable salts thereof. The definition of the compounds of formula (Ia) and (Ib) is therefore also intended to encompass the chemical structures of all R- and S-isomers in any ratio, for example, with an enrichment (i.e., enantiomeric excess or diastereomeric excess) of one of the possible isomers, and a corresponding smaller ratio of the other isomer. In the particular case of amino acids, they may acquire the L or D configuration.

[0077] The compounds of formula (Ia) and (Ib) may be provided in any form suitable for the intended administration, including in particular pharma- ceutically acceptable salts of the compounds of formula (Ia) and (Ib).

[0078] Pharmaceutically acceptable salt refers to salts of the compounds of formula (Ia) and (Ib) that are considered acceptable for clinical, veterinary and / or cosmetic use. Exemplary pharmaceutically acceptable salts include salts prepared by reaction of the compounds of formula (Ia) and (Ib) with mineral or organic acids or organic or inorganic bases. Such salts are known as acid addition salts and base addition salts, respectively. It will be recognized that the nature of the particular counterion or counterions forming part of any salt is not critical, so long as the salt as a whole is pharmaceutically acceptable and the counterion does not impart undesirable properties to the salt as a whole. These salts can be prepared by methods known to those skilled in the art.

[0079] The term "pharmaceutically, cosmetically, or diagnostically acceptable salt" includes commonly used non-toxic salts. The preparation of pharma- ceutically, cosmetically, or diagnostically acceptable salts of the compounds of the present invention can be carried out by methods well known in the art. In general, such salts can be prepared by reacting the free acid or base form of the compounds of the present invention with a stoichiometric amount of the appropriate base or acid, respectively, in a suitable solvent such as water, an organic solvent, or a mixture thereof.

[0080] Examples of pharma- ceutically acceptable addition salts include those with inorganic acids, such as hydrochloric, hydrobromic, sulfuric, nitric, hydroiodic, metaphosphoric, or phosphoric acid, as well as with organic acids, such as succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, trifluoroacetic, malic, lactic, formic, propionic, glycolic, gluconic, camphorsulfuric, isothionic, mucic, gentisic, isonicotinic, saccharic, glucuronic, furoic, glutamic, ascorbic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), ethanesulfonic, and tert-Butyl acids. and arylsulfonic acids, such as benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, methanesulfonic acid or naphthalenesulfonic acid; base addition salts formed with alkali metals and alkaline earth metals, such as N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), lysine and procaine, as well as internally formed salts.

[0081] As used herein, the term "labeling or imaging" refers to a molecule that facilitates the visualization and / or detection of the targeting molecule disclosed herein. In the context of this disclosure, the expression "labeling agent or imaging agent" refers to any substance that is used as a label or enhances a specific structure in any imaging technique. Imaging agents therefore include optical imaging agents, magnetic resonance imaging agents, radioisotopes, and contrast agents. Imaging agents or labeling agents are well known in the art. Specific examples of imaging agents or labeling agents are gases such as sterile air, oxygen, argon, nitrogen, fluorine, perfluorocarbons, carbon dioxide, nitrogen dioxide, xenon and helium; commercially available agents used in positron emission tomography (PET), computer-assisted tomography (CAT), single photon emission computed tomography, X-ray, fluoroscopy, and magnetic resonance imaging (MRI). Examples of materials suitable for use as contrast agents in MRI include currently available gadolinium chelates such as diethylenetriaminepentaacetic acid (DTPA) and gadopentetate dimeglumine, as well as iron, magnesium, manganese, copper, and chromium. Examples of materials useful for CAT and X-ray include iodine-based materials for intravenous administration, ionic monomers such as diatrizoate and iothalamate, non-ionic monomers such as iopamidol, isohexol, and ioversol, non-ionic dimers such as iotrol and iodixanol, ionic dimers such as ioxagalte. Other useful materials include insoluble salts such as barium and zinc acetate for oral use. In some molecules, the imaging agent is a dye. In some molecules, the imaging agent is a fluorescent moiety. In some molecules, the fluorescent moiety is selected from fluorescent proteins, fluorescent peptides, fluorescent dyes, fluorescent substances, or combinations thereof.Examples of fluorescent dyes include, but are not limited to, xanthenes (e.g., rhodamine, rhodol, fluorescein, and their derivatives); bimanes; coumarins and their derivatives (e.g., umbelliferone and aminomethylcoumarin); aromatic amines (e.g., dansyl, squarate dyes); benzofurans; fluorescent cyanines; indocarbocyanines; carbazoles; dicyanomethylenepyrans; polymethines; oxabenzanthranes; xanthenes; pyryliums; carbostils; perylenes; acridones; quinacridones; rubrenes; anthracenes; coronenes; phenanthrecenes; pyrenes; butadienes; stilbenes; porphyrins; phthalocyanines; lanthanide metal chelate complexes; rare earth metal chelate complexes; and derivatives of such dyes. Examples of fluorescein dyes include, but are not limited to, 5-carboxyfluorescein, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate and 6-carboxyfluorescein. Examples of rhodamine dyes include, but are not limited to, tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxyrhodol derivatives, tetramethyl and tetraethylrhodamine, diphenyldimethyl and diphenyldiethylrhodamine, dinaphthylrhodamine, rhodamine 101 sulfonyl chloride (sold under the trade name TEXAS RED®). Examples of cyanine dyes include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, IRDYE680, AlexaFluor750, IRDye800CW, ICG. Examples of fluorescent peptides include GFP (Green Fluorescent Protein) or derivatives of GFP (e.g., EBFP, EBFP2, Azurite, mKalama1, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, YPet). The fluorescent label is detected by any suitable method.For example, fluorescent labels can be detected by exciting the fluorescent dye with light of an appropriate wavelength and detecting the resulting fluorescence, for example, by microscopy, visual inspection, photographic film, or by using electronic detectors such as charge-coupled devices (CCDs), photomultipliers, etc. In some molecules, imaging agents are labeled with positron-emitting isotopes (e.g., 18F) for positron emission tomography (PET), gamma-ray isotopes (e.g., 99mTc) for single-photon emission computed tomography (SPECT), or paramagnetic molecules or nanoparticles (e.g., Gd3+ chelates or coated magnetite nanoparticles) for magnetic resonance imaging (MRI). In some molecules, imaging agents are labeled with gadolinium chelates, iron oxide particles, superparamagnetic iron oxide particles, ultrasmall paramagnetic particles, manganese chelates, or gallium-containing agents. Examples of gadolinium chelates include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA). In some molecules, the imaging agent is a near-infrared fluorophore for near-infrared (near-IR) imaging, luciferase (firefly, bacteria, or coelenterate) or other luminescent molecules for bioluminescence imaging, or perfluorocarbon-filled vesicles for ultrasound. In some molecules, the imaging agent is a nuclear probe. In some molecules, the imaging agent is a SPECT or PET radionuclide probe. In some molecules, the radionuclide probe is selected from technetium chelates, copper chelates, radioactive fluorides, radioactive iodines, and indium chelates. Examples of Tc chelates include, but are not limited to, HYNIC, DTPA, and DOTA. In some molecules, the imaging agent comprises a radioactive moiety as described by Lu et al., e.g., a radioisotope such as 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 64Cu radioisotope.

[0082] The term "cell targeting agent" refers to any biological or chemical structure that exhibits an affinity for a molecule present in the human or animal body, which can direct functionalized nanoparticles by directing them to the target site of therapeutic treatment, for example because it selectively binds to a receptor expressed or overexpressed on a particular cell type. The term thus includes ligands for a particular receptor or antigen, for example, an antibody for a particular antigen, folic acid for its receptor, or a sugar such as galactose for a hepatic receptor. The targeting agent can be attached to the functionalized end group of the anionic polymer via the A and / or A' moieties, or it can also be attached to the cationic polymer.

[0083] Cell targeting groups are well known in the art. Examples of targeting agents include, but are not limited to, monoclonal and polyclonal antibodies (e.g., IgG, IgA, IgM, IgD, IgE antibodies), sugars (e.g., mannose, mannose-6-phosphate, galactose, galactosamine, mannosamine), proteins (e.g., transferrin), oligopeptides (e.g., cyclic and acyclic RGD-containing oligopeptides), oligonucleotides (e.g., aptamers), and vitamins (e.g., folate), Her-2 binding peptides, TLR agonists, β-D-glucose, Asn-Gly-Arg peptides, angiopep2, aptamers (A-9, A10, anti-gp 120, TTA1, sgc8, anti-MUC-1, AS1411), primaquine, zidovudine, superoxide dismutase, prednisolone, platinum, cisplatin, sulfamethoxazole, amoxicillin, etoposide, mesalidine, doxorubicin, paclitaxel, 5-aminosalicylic acid, denosumab, docetaxel, calcitonin, proanthocyanidins, methotrexate, camptothecin, galactose, glycyrrhetinic acid, lactose, hyaluronic acid, octeotolide, lactobionic acid, β-galactosyl moiety, arabino-galactan, chitosan, azo-based polyphosphazenes, azo groups and 4-amino-benzyl-carbamate, succinic acid, 4,4'-Dihydroxyazobenzene-3-carboxylic acid, cyclic RGD pentapeptide, aspartic acid octapeptide, alendronate, transferrin, bisphosphonate adendrone, monosialoganglioside GM1, glutathione, E-selectin thioaptamer, poloxamer-407, urokinase-type plasminogen activator receptor (uPAR) antagonist, CXCR4 chemokine receptor antagonist, GRP78 peptide antagonist, RGD peptide, RGD cyclic peptide, luteinizing hormone releasing hormone (LHRH) antagonist peptide, aminopeptidase targeting peptide, brain homing peptide, kidney homing peptide, heart homing peptide, gut homing peptide, integrin homing peptide, angiogenic tumor endothelium homing peptides, ovary homing peptides, uterus homing peptides, sperm homing peptides, microglia homing peptides, synovial membrane homing peptides, urothelium homing peptides, prostate homing peptides, lung homing peptides (RCPLSHSLICY), laminin receptor binding peptides (e.g., YIGSR), skin homing peptides, retina homing peptides, pancreatic homing peptides, liver homing peptides, lymph node homing peptides, adrenal gland homing peptides, thyroid homing peptides, bladder homing peptides, breast homing peptides, neuroblastoma homing peptides, lymphoma homing peptides, muscle homing peptides, wound vasculature homing peptides, adipose tissue homing peptides, virus binding peptides, or fusogenic peptides.

[0084] In a particular embodiment, in the anionic polymer defined herein, m is an integer selected from 5 to 250, preferably 20 to 160, more preferably 30 to 150, even more preferably 50 to 140, and particularly preferably 60 to 125; and n is an integer selected from 3 to 200, preferably 4 to 100, more preferably 4 to 40, preferably 5 to 30, more preferably 6 to 26, and particularly preferably 8 to 20.

[0085] In certain embodiments, in the anionic polymers defined herein, the ratio of m:n is in the range of 1:8 to 30:1, preferably 1:5 to 10:1, more preferably 1:1 to 8:1, even more preferably 2:1 to 7:1.

[0086] In a particular embodiment, in the anionic polymer defined herein, m is an integer selected from 5 to 250, preferably 20 to 160, more preferably 30 to 150, even more preferably 50 to 140, and particularly preferably 60 to 125; and n is an integer selected from 3 to 200, preferably 4 to 100, more preferably 4 to 40, preferably 5 to 30, more preferably 6 to 26, and particularly preferably 8 to 20; with the proviso that the ratio of m:n is in the range of 1:8 to 30:1, preferably 1:5 to 10:1, more preferably 1:1 to 8:1, and even more preferably 2:1 to 7:1.

[0087] According to certain embodiments, the anionic polymer is of formula (Ia) or (Ib), where R3 is -CH3 and R2 is H.

[0088] According to a particular embodiment, the anionic polymer is of formula (Ia) or (Ib), in which R3 is -CH3 and R2 is H; m is an integer selected from 5 to 250, preferably from 20 to 160, more preferably from 30 to 150, even more preferably from 50 to 140, and particularly preferably from 60 to 125; n is an integer selected from 3 to 200, preferably from 4 to 100, more preferably from 4 to 40, preferably from 5 to 30, more preferably from 6 to 26, and particularly preferably from 8 to 20; with the m:n ratio being in the range of 1:8 to 30:1, preferably from 1:5 to 10:1, more preferably from 1:1 to 8:1, and even more preferably from 2:1 to 7:1.

[0089] According to certain embodiments, the anionic polymer is of formula (Ia) or (Ib), R3 is H; and R2 is selected from the group consisting of (X) and (XII).

[0090] According to a particular embodiment, the anionic polymer is of formula (Ia) or (Ib), wherein R3 is H; R2 is selected from the group consisting of (X) and (XII); m is an integer selected from 5 to 250, preferably 20 to 160, more preferably 30 to 150, even more preferably 50 to 140, particularly preferably 60 to 125; n is an integer selected from 3 to 200, preferably 4 to 100, more preferably 4 to 40, preferably 5 to 30, more preferably 6 to 26, particularly preferably 8 to 20; with the m:n ratio ranging from 1:8 to 30:1, preferably 1:5 to 10:1, more preferably 1:1 to 8:1, even more preferably 2:1 to 7:1.

[0091] Some preferred examples of anionic polymers according to formula (Ib) are according to formula (Ib1) shown below, where Z is a single bond. [ka]

[0092] In certain embodiments, the anionic polymer is of formula (Ib1); R2 is selected from the group consisting of radicals selected from the group consisting of (X), (XI), (XII), and (XIII).

[0093] In a particular embodiment, the anionic polymer is of formula (Ib1), wherein R2 is selected from the group consisting of radicals selected from the group consisting of (X), (XI), (XII), and (XIII); m is an integer selected from 5 to 250, preferably 20 to 160, more preferably 30 to 150, even more preferably 50 to 140, and particularly preferably 60 to 125; n is an integer selected from 3 to 200, preferably 4 to 100, more preferably 4 to 40, preferably 5 to 30, more preferably 6 to 26, and particularly preferably 8 to 20; with the m:n ratio being in the range of 1:8 to 30:1, preferably 1:5 to 10:1, more preferably 1:1 to 8:1, and even more preferably 2:1 to 7:1.

[0094] In another particular embodiment, the anionic polymer is of formula (Ib1);

[0095] In another particular embodiment, the anionic polymer is of formula (Ib1); where R2 is H and R3 is -CH3.

[0096] In a preferred embodiment, the anionic polymer is of formula (Ib1), where R2 is H and R3 is -CH3; m is an integer selected from 5 to 250, preferably 20 to 160, more preferably 30 to 150, even more preferably 50 to 140, and particularly preferably 60 to 125; n is an integer selected from 3 to 200, preferably 4 to 100, more preferably 4 to 40, preferably 5 to 30, more preferably 6 to 26, and particularly preferably 8 to 20; with the proviso that the ratio of m:n is in the range of 1:8 to 30:1, preferably 1:5 to 10:1, more preferably 1:1 to 8:1, and even more preferably 2:1 to 7:1.

[0097] When present, the purpose of the R5 moiety is to simulate or mimic the activity of GALA peptide (and analogues), which is well known as a tool for enhancing transfection activity, more specifically to enhance cellular uptake, membrane permeation and endosomal escape of active agents, and to improve cytosolic delivery of active agents. All of these result in a significant improvement in the efficiency of the transcription / translation process. GALA peptides have been shown to promote cellular internalization through fragmentation, membrane fusion and pore formation as well as membrane binding properties. Considering the important role of internalization and endosomal escape mechanisms for the effective delivery of a given cargo, the approach described herein is also based on non-covalent coating of GALA shielding polymers on the nanoparticle surface. Upon contact with the cell membrane, GALA-derived polymers may detach from the nanoparticle surface and insert into the membrane by hydrophobic interactions to form pores that allow endosomal scape or cytosolic delivery of nanoparticle cargo or proteins.

[0098] In a preferred embodiment, the anionic polymer is of formula (Ib), wherein Z is selected from the group consisting of -CO-(CH2)qSS-(CH2)r-NH-(R5)z-, -CO-(CH2)p-CO-NH-(CH2)qSS-(CH2)r-NH-(R5)z-, -CO-(CH2)p-NH-(R5)z-, and -(R5)z-; wherein R5 is a random or block copolymer comprising at least two different repeat units selected from the group consisting of (II), (III), (IV), (V), (VI), (VII), (VIII), and (IX); wherein the repeat units of each of formulas (II), (III), (IV), (V), (VI), (VII), (VIII), and (IX) in R5 and the sequential order of the repeat units indicated in the square brackets with the integer n may be present in blocks or randomly.

[0099] According to certain embodiments, the anionic polymer is of formula (Ib), where Z is selected from the group consisting of -CO-(CH2)qSS-(CH2)r-NH-(R5)z-, -CO-(CH2)p-CO-NH-(CH2)qSS(CH2)r-NH-(R5)z-, -CO-(CH2)p-NH-(R5)z-, and -(R5)z-; R5 is at least two different repeating groups selected from the group consisting of (III), (VI), (VIII), and (IX). wherein c, e, g, and h are integers independently selected from 0 to 20, with the proviso that at least one, preferably at least two, and more preferably at least three of c, e, g, and h are different from 0; z is an integer selected from 5 to 35; wherein the sequential order of each repeat unit of R5 and the repeat units indicated in the square brackets with the integer n may be present in blocks or randomly.

[0100] According to certain embodiments, the anionic polymer is of formula (Ib), where Z is selected from the group consisting of -CO-(CH2)qSS-(CH2)r-NH-(R5)z-, -CO-(CH2)p-CO-NH-(CH2)qSS(CH2)r-NH-(R5)z-, -CO-(CH2)p-NH-(R5)z-, and -(R5)z-; R5 is a random or block copolymer comprising at least two different repeat units selected from the group consisting of (III), (VI), (VIII), and (IX); where c, e, g, and h are integers independently selected from 0 to 20, with the proviso that at least one, preferably at least two, and more preferably at least three of c, e, g, and h are z is different from 0; m is an integer selected from 5 to 250, preferably 20 to 160, more preferably 30 to 150, even more preferably 50 to 140, and particularly preferably 60 to 125; n is an integer selected from 3 to 200, preferably 4 to 100, more preferably 4 to 40, preferably 5 to 30, more preferably 6 to 26, and particularly preferably 8 to 20; provided that the ratio of m:n is in the range of 1:8 to 30:1, preferably 1:5 to 10:1, more preferably 1:1 to 8:1, and even more preferably 2:1 to 7:1; wherein the consecutive order of the repeat units of R5 and the repeat units indicated in the square brackets with the integer n may be present in blocks or randomly.

[0101] Some preferred examples of anionic polymers according to formula (Ib) are according to formula (Ib2) shown below, where Z is (R5)z: [ka] wherein b, e, f, g, and h are each independently an integer selected from 0 to 20; and wherein at least two, more preferably at least three, of b, e, f, g, and h are different from 0; z is an integer selected from 5 to 100, preferably 5 to 35; m is an integer selected from 5 to 250, preferably 20 to 160, more preferably 30 to 150, even more preferably 50 to 140, and particularly preferably 60 to 125; n is an integer selected from 3 to 200, preferably 4 to 100, more preferably 4 to 40, preferably 5 to 30, more preferably 6 to 26, and particularly preferably 8 to 20; with the m:n ratio being in the range of 1:8 to 30:1, preferably 1:5 to 10:1, more preferably 1:1 to 8:1, and even more preferably 2:1 to 7:1; where the " / " between bracketed repeat units with integers b, e, n, f, g, and h indicates that the sequential order of the bracketed monomer repeat units on either side of the symbol is arbitrary, i.e., they can be present in any order, and they can be present in blocks or randomly; Here, the " / " between a bracketed repeat unit with an integer m and a bracketed repeat unit with an integer z+n indicates that the sequential order of the monomer repeat units on either side of the symbol is arbitrary, i.e., they can be present in any order, and they can be present in blocks or randomly.

[0102] According to a particular embodiment, the anionic polymer is of formula (Ia); R2 is a radical selected from the group consisting of H and (X) and (XII).

[0103] In another particular embodiment, the anionic polymer is of formula (Ia); R2 is selected from H and a radical selected from the group consisting of (X) and (XII); and R3 is -CH3.

[0104] In a preferred embodiment, the anionic polymer is of formula (Ia); in which R2 is H and a radical selected from the group consisting of (X) and (XII), and R3 is -CH3; m is an integer selected from 5 to 250, preferably 20 to 160, more preferably 30 to 150, even more preferably 50 to 140, and particularly preferably 60 to 125; n is an integer selected from 3 to 200, preferably 4 to 100, more preferably 4 to 40, preferably 5 to 30, more preferably 6 to 26, and particularly preferably 8 to 20; with the m:n ratio being in the range of 1:8 to 30:1, preferably 1:5 to 10:1, more preferably 1:1 to 8:1, and even more preferably 2:1 to 7:1.

[0105] In a preferred embodiment, the anionic polymer is of formula (Ia), in which Y is -CO-(CH2)p-CO-; R2 is H and a radical selected from the group consisting of (X) and (XII); R3 and R4 are H; m is an integer selected from 5 to 250, preferably 20 to 160, more preferably 30 to 150, even more preferably 50 to 140, and particularly preferably 60 to 125; n is an integer selected from 3 to 200, preferably 4 to 100, more preferably 4 to 40, preferably 5 to 30, more preferably 6 to 26, and particularly preferably 8 to 20; with the m:n ratio being in the range of 1:8 to 30:1, preferably 1:5 to 10:1, more preferably 1:1 to 8:1, and even more preferably 2:1 to 7:1.

[0106] Examples of cationic polymers include poly-L-lysine (PLL), poly-L-ornithine (PLO), poly-L-histidine, polyamidoamines, polyarginines, poly-[2-{(2-aminoethyl)aminoethyl-aspartamide] (pAsp(DET)), poly(dimethylaminoethyl methacrylate) (pDMAEMA), polyethyleneimine (PEI), chitosan, poly(beta amino esters), cationic or cationically ionizable lipids or lipid-like substances, block, random or graft polycations based on polyamino acids. and combinations of anionic polymers, such as block copolymers of polyethylene glycol and polyarginine, block copolymers of polyethylene glycol and polylysine, block copolymers of polyethylene glycol and poly-[amino acid-based, such as block copolymers of 2-{(2-aminoethyl)amino}]-ethyl-aspartamide] (PEG-pAsp(DET)), and any other suitable cationic polymer, so long as the polymer is capable of forming a polymer complex with at least one anionic polymer of formula (Ia) or (Ib) of the present invention.

[0107] According to some embodiments, at least one anionic polymer of formula (Ia) or (Ib) of the present invention electrostatically interacts with positively charged proteins and acts as a shielding layer on top of the protein or is intercalated into the protein.

[0108] Thus, according to some embodiments, a polymer complex or a protein-based complex as defined herein may comprise one or more anionic polymers of formula (Ia) or (Ib). According to some embodiments, a polymer complex or a protein-based complex comprises at least two different anionic polymers of formula (Ia) or (Ib) as defined herein.

[0109] According to some embodiments, the anionic polymer comprises an R5 moiety as defined in claim 1, which may be present in blocks or randomly in combination with repeat units indicated in square brackets with an integer n.

[0110] Some examples according to this embodiment, in which the anionic polymer is of formula (Ib) and includes R5 moieties that may be present in blocks or randomly in combination with repeat units indicated in square brackets with an integer n, may exhibit particular endosomolytic activity.

[0111] In certain embodiments, the endosomolytic moiety has its active conformation at endosomal pH (e.g., pH 5-6). An "active" conformation is one in which the endosomolytic ligand promotes lysis of an endosome and / or transport of a modular composition of the invention from an endosome to the cytoplasm of a cell.

[0112] As described above, the present invention provides a) a cationic polymer covalently or electrostatically associated with at least one active agent, preferably selected from the group consisting of pharmaceutical active agents, veterinary active agents, cosmetic active agents, diagnostic active agents, nucleic acids, peptides, antibodies, aptamers, proteins, and mixtures thereof, wherein the cationic polymer and the at least one active agent form positively charged nanoparticles; b) at least one anionic polymer of formula (Ia) or (Ib) as defined herein, which interacts electrostatically with the nanoparticles; The present invention relates to a polymer complex comprising:

[0113] The skilled artisan knows that different nanoparticle systems can be used to load the active ingredient, which may be in the form of micelles, cylindrical micelles, reverse micelles, vesicles, lipid-polymer hybrid nanoparticles or liposomes.

[0114] According to another aspect of the present invention, a) a positively charged protein; b) at least one anionic polymer of formula (Ia) or (Ib) as defined herein, which interacts electrostatically with the protein; A protein-based complex comprising:

[0115] According to these aspects of the invention, at least one anionic polymer of formula (Ia) or (Ib) acts as a shielding layer on top of or is intercalated between nanoparticles or positively charged proteins.

[0116] The at least one active agent(s) may be covalently attached directly or through one or more linkers, or the at least one active agent(s) may be non-covalently attached to the compound.

[0117] In a preferred embodiment, at least one active agent is covalently linked to the polypeptide backbone of the cationic polymer via an amino acid side residue, C- or N-terminal group via an amide, ester, anhydride bond, or via a linker containing one or more functional groups, including, but not limited to, alkyne, azide, reactive disulfide, maleimide, hydrazide, hydrazone, Schiff base, acetal, aldehyde, carbamate, and reactive ester. In an alternative embodiment, the covalent linkage is bioresponsive.

[0118] In another preferred embodiment, at least one active agent is linked to the polypeptide backbone of the cationic polymer via electrostatic interactions. Examples of anionic compounds include proteins, polysaccharides, small molecules, and nucleic acids.

[0119] The conditions for preparation, such as the aqueous medium, pH, temperature, ionic strength, etc., can be appropriately adjusted by those skilled in the art.

[0120] According to a particular embodiment, the polymer complex is obtained upon mixing in an aqueous medium at a pH in the range of 4 to 9, preferably in the range of 4.5 to 8.5, more preferably in the range of 5 to 7.5, and particularly preferably in the range of 6.5 to 7.4. The pH can be easily adjusted using a buffer as the solvent.

[0121] According to a specific embodiment, the ionic strength of the solution to be mixed can be appropriately adjusted within a range that does not destroy the structure of the nanoparticles or inhibit the inclusion of the substance to be encapsulated in the nanoparticles, and is preferably within the range of 0 to 1000 mM, preferably 0 to 300 mM, more preferably 0 to 150 mM, and particularly preferably 0 to 50 mM.

[0122] According to a particular embodiment, the average molecular weight (Mw) of the compound of formula (Ia) or (Ib) according to the present invention is in the range of 200 Da to 80,000 Da, preferably 500 Da to 60,000 Da, more preferably 2,000 Da to 40,000 Da, more preferably 2,500 Da to 30,000 Da, as measured by gel permeation chromatography-refractive index-multi-angle light scattering-visible-ultraviolet (GPC-RI-MALS-UV).

[0123] According to certain embodiments, the at least one active agent is selected from the group consisting of low molecular weight drugs, peptides, antibodies, hormones, enzymes, nucleic acids, proteins, and combinations thereof.

[0124] According to certain embodiments, the polymer complex (also referred to herein as a polyplex) comprises at least one nucleic acid. In certain embodiments, the polymer complex comprises a combination of two or more nucleic acids.

[0125] As used herein, the term "nucleic acid" refers to DNA or RNA.In certain embodiments, nucleic acid is DNA / RNA hybrid, short interfering RNA (siRNA), microRNA (miRNA), single-stranded RNA (sgRNA), donor DNA, self-amplifying / replicating RNA, circular RNA (oRNA), plasmid DNA (pDNA), closed linear DNA (clDNA), short hairpin RNA (shRNA), messenger RNA (mRNA), and antisense RNA (aRNA), messenger RNA (mRNA), CRISPR guide RNA, antisense nucleic acid, decoy nucleic acid, aptamer, and ribozyme, to name a few, and includes both nucleotide sequence and any structural aspect thereof, such as double-stranded, single-stranded, helical, hairpin, and may include modified or unmodified bases.

[0126] When separate nucleic acids are provided, they may be all DNA molecules or all RNA molecules, or may be molecules that contain a mixture of DNA and RNA molecules or an association of DNA and RNA strands.

[0127] The nucleic acid may be a poly or oligonucleotide, such as an oligo or poly double-stranded RNA, an oligo or poly double-stranded DNA, an oligo or poly single-stranded RNA, an oligo or poly single-stranded DNA, etc. Each of the nucleotides contained in the nucleic acid may be a naturally occurring nucleotide or a chemically modified non-natural nucleotide.

[0128] The length of the nucleic acid is not particularly limited, and the nucleic acid may have a short chain in the range of 10 to 200 bases, preferably 20 to 180 bases, preferably 25 to 100 bases, preferably 30 to 50 bases; or the nucleic acid may have a relatively long chain of 200 to 20,000 bases, more preferably 250 to about 15,000 bases.

[0129] According to certain embodiments, the nucleic acid is a closed linear DNA (clDNA), i.e. a molecule in which the double-stranded region is flanked and protected by two single-stranded loops, thereby generating a dumbbell-shaped molecule.

[0130] In a more particular embodiment, the clDNA consists of a stem region comprising a double-stranded DNA sequence of interest covalently closed at both ends by hairpin loops, wherein the clDNA comprises at least two modified nucleotides.

[0131] As used herein, the term "closed linear DNA" or "clDNA" refers to a covalently closed single-stranded DNA molecule that forms a "dumbbell" or "doggybone" shaped structure under conditions that allow nucleotide hybridization. Thus, clDNA is formed by a single-stranded DNA molecule, but the formation of the "dumbbell" structure by hybridization of two complementary sequences within the same molecule produces a structure consisting of a double-stranded intermediate segment flanked by two single-stranded loops. Those skilled in the art will recognize how to generate clDNA from open or closed double-stranded DNA using routine molecular biology techniques. For example, those skilled in the art will recognize that clDNA can be generated by attaching a hairpin DNA adaptor to both ends of an open double-stranded DNA, for example, by the action of a ligase. A "hairpin DNA adaptor" refers to a single-stranded DNA that forms a stem-loop structure by hybridization of two complementary sequences, where the stem region formed is closed at one end by a single-stranded loop and open at the other end.

[0132] A "modified nucleotide" is any nucleotide (e.g., adenosine, guanosine, cytidine, uracil, and thymidine) that has been chemically modified by modification of the base, sugar, or phosphate group, or that incorporates a non-natural moiety into its structure. Thus, modified nucleotides can be naturally occurring or non-naturally occurring, depending on the modification.

[0133] The polymer complexes or protein-based complexes of the present disclosure constitute useful tools for therapeutic or diagnostic indications, where the compounds of formula (Ia) or (Ib) as defined herein act as a protective shield for the positively charged nanoparticles or positively charged proteins carrying the active ingredient, leading to improved specific properties such as extended circulation time, safety or toxicological profile, or release profile in physiological conditions, and in the case of the polymer complexes also improved transfection efficiency into the desired cells.

[0134] The polymer complex may have a particle hydrodynamic diameter in the range of 10 nm to 2000 nm, preferably 20 nm to 800 nm, more preferably 25 nm to 350 nm, 30 nm to 300 nm, 30 nm to 200 nm, as measured by a dynamic light scattering instrument.

[0135] The protein-based complexes may have a particle hydrodynamic diameter in the range of 2 nm to 2000 nm, preferably 5 nm to 1000 nm, more preferably 10 nm to 800 nm, 15 nm to 700 nm, 20 nm to 600 nm, as measured by a dynamic light scattering instrument. A further aspect of the present disclosure relates to a pharmaceutical, diagnostic or therapeutic composition comprising at least one polymer complex or at least one protein-based complex as defined above, together with one or more suitable pharma- ceutically or diagnostically acceptable excipients.

[0136] A further aspect of the present disclosure relates to a polymer complex, protein-based complex, or composition of the present disclosure for use as a pharmaceutical, in diagnostics or theranostics.

[0137] This aspect of the disclosure can be reformulated as the use of a polymer complex, a protein-based complex, or a pharmaceutical composition of the disclosure to manufacture a medicament.

[0138] This embodiment can also be formulated as a method for the treatment, diagnosis, prevention and / or theranostics of disease, which method comprises administering to a subject, including a human, in need thereof a therapeutically, diagnostically, prophylactically and / or theranostically effective amount of a polymer complex, protein-based complex, or composition of the invention, together with one or more suitable pharma- ceutical , veterinary or cosmetically acceptable excipients and / or carriers.

[0139] In the present invention, the "subject" may be a mammal, including a human. The subject may be a healthy subject or a subject suffering from any disease.

[0140] In the present invention, "treatment" refers to curing, preventing or inducing remission of a disease or disorder, or slowing the rate of progression of a disease or disorder. Treatment can be achieved by administering a therapeutically effective amount of a pharmaceutical composition.

[0141] When the method refers to diagnosis, this embodiment can also be formulated as a method for diagnosing disease in an isolated sample of a subject, the method comprises administering an effective amount of any of the polymer complexes to said subject or to an isolated sample of a subject with a pharmaceutical composition having one or more imaging agents as defined above.The detection of these imaging agents can be performed by well-known techniques, such as imaging diagnostic techniques.Examples of imaging diagnostic techniques suitable for the present disclosure include, but are not limited to, ultrasound imaging, magnetic resonance imaging (MRI), fluoroscopy, X-ray, positron emission tomography (PET), single photon emission computed tomography (SPECT), fluorescence microscopy, and in vivo fluorescence.

[0142] Thus, the present disclosure also relates to the use of the polymer complexes, or pharmaceutical compositions of the present disclosure as bioimaging tools, particularly for tracking the internalization and delivery of active or imaging agents.

[0143] "Bioimaging tools" are to be understood by this description as reagents used in imaging techniques used in biology to track cells or certain compartments of a particular tissue. Examples of bioimaging tools include chemiluminescent compounds, fluorescent and phosphorescent compounds, compounds that emit X-rays or alpha, beta or gamma rays, etc.

[0144] A further aspect of the present disclosure relates to the use of the polymer complexes defined herein as non-viral vectors commonly used in biomedical applications, such as vaccines or gene therapy, which are effective for transfection of host eukaryotic cells in culture, in vivo or ex vivo, unicellular parasites and bacteria, including gene editing using the CRISPR / Cas9 approach.

[0145] A further aspect of the present disclosure relates to the use of a protein-based complex as defined herein as a carrier for general use in protein-based therapeutics, such as vaccines.

[0146] In a particular embodiment, the present invention relates to the use of the polymer complexes defined herein as transfection reagents for delivering active agents (preferably nucleic acids, circular and linear nucleic acids, regardless of size and structure) to target cells in vivo, in vitro or ex vivo. In a particular embodiment, the active agent is selected from the group consisting of low molecular weight drugs, peptides, proteins, antibodies, nucleic acids, aptamers, and combinations thereof.

[0147] The above transfection reagents are also useful for co-transfection of two or more active agents simultaneously, for example, two or more nucleic acids.

[0148] Transfection compositions (such as kits) and methods of using the transfection reagents to deliver nucleic acids to target cells are also within the scope of the invention. Further embodiments will be apparent upon review of this disclosure.

[0149] The present invention also relates to methods for delivering active agents in vitro, ex vivo, and in vivo that involve using the polymer complexes disclosed herein.

[0150] The present invention also provides the composition for use as a pharmaceutical composition to induce a modulatory effect on the expression of one or more target proteins that cause or are involved in genetic inherited or complex genetic diseases, immune diseases, cancer, viral infections in various tissues / organs, or tumors.

[0151] The present invention also relates to the in vitro or ex vivo use of the composition according to the present invention in the manufacture of biological products, in particular biological products that code recombinant protein, peptide or antibody; or in the production of recombinant viruses, such as adeno-associated virus (AAV), lentivirus (LV), adenovirus, oncolytic virus, baculovirus, or virus or virus-like particle, the composition comprises a polymer complex as defined herein and comprises at least one nucleic acid molecule for transfection.The term "biological product" as used herein refers to protein or nucleic acid or their combination, biological entities such as cells or viruses, cell compartments, organoids, and tissues.

[0152] The present invention also relates to the in vitro or ex vivo use of the polymer complexes according to the invention for genome engineering, for cell reprogramming, for cell differentiation or gene editing.

[0153] Compositions for transfecting cells include a polymer complex as defined herein and an acceptable excipient, buffer, cell culture medium, or transfection medium.

[0154] The present invention also relates to the composition defined herein for use as a therapeutic or preventive vaccine against viral infection, or as a therapeutic vaccine against cancer.Generally, in this aspect, the vaccine is delivered by direct administration, such as systemic administration, intramuscular administration, intradermal administration, intraperitoneal administration, intratumoral administration, oral administration, topical administration, or subcutaneous administration, in which the composition is combined with a pharmaceutically acceptable vehicle.In other words, the vaccine can be directly injected into the body, particularly into a human individual, to induce cellular and / or humoral response.

[0155] Cellular targeting is achieved through a variety of mechanisms and depends on the nature and characteristics of the transfection reagent, the composition or formulation of the method or protocol, and the route of administration.

[0156] In a more particular embodiment, the present invention relates to a polymer complex for use in the prevention and / or treatment of various diseases such as neurodegenerative diseases, neurological diseases, cancer, infectious diseases, ageing-related diseases, neuroinflammation, demyelinating diseases, multiple sclerosis, ischemic disorders, immune disorders, inflammatory disorders, rare diseases, among others, depending on the active agent it carries.

[0157] The compounds described in the present disclosure, their pharma- ceutically acceptable salts and solvates, and pharmaceutical compositions containing them, can be combined with other additional drugs to provide combination therapy.These additional drugs can be part of the same pharmaceutical composition or can be provided in the form of separate compositions for simultaneous or non-simultaneous administration with the pharmaceutical composition containing the compound of formula (I), its pharma- ceutically acceptable salts, stereoisomers, or solvates.

[0158] The compounds of the present disclosure may be in crystalline form as free form or as solvates, and both forms are intended to be included within the scope of the present disclosure. In this respect, the term "solvate" as used herein includes both pharmaceutically acceptable solvates, i.e., solvates of the compounds of formula (I) that can be used in the preparation of pharmaceuticals, and pharmaceutically unacceptable solvates, which may be useful in the preparation of pharmaceutically acceptable solvates or salts. The nature of the pharmaceutically acceptable solvate is not important, so long as it is pharmaceutically acceptable. In certain embodiments, the solvate is a hydrate. The solvates can be obtained by conventional solvation methods well known to those skilled in the art. Unless otherwise stated, the compounds of the present disclosure also include compounds that differ only in the presence of one or more isotopically enriched atoms. Examples of isotopically enriched atoms are, but are not limited to, deuterium, tritium, 13C or 14C, or nitrogen atoms enriched with 15N.

[0159] One of ordinary skill in the art will recognize that monomeric repeat units are defined by brackets ("[]") drawn around the repeating monomeric unit. The number (or letter representing a numerical range) to the right of the brackets represents the number of monomeric repeat units present in the polymer chain.

[0160] In the context of the present disclosure, the term "polyplex" or "polymer complex" refers to a compound formed by electrostatic interactions between a cationic polymer as described herein and at least one polyanionic genetic material (preferably a nucleic acid). The cationic polymer contains an opposite charge to the polyanionic genetic material at a given pH, resulting in the formation of multiple electrostatic bonds between the polyanionic genetic material and the polymer at a given pH. The driving force for polymer complex formation is the multivalency of both the polyanionic nucleic acid and the polycationic polymer, which results in highly effective entropically driven condensation of the genetic material. Polymer complexes (polyplexes) containing nucleic acids are useful as non-viral synthetic vectors that can deliver nucleic acids to target cells. DNA or RNA delivery to target cells by non-viral synthetic vectors (such as polyplexes) has been widely recognized as a promising alternative delivery method to using viral vectors, which face significant challenges and drawbacks. These include immunogenic responses (which may prevent re-administration), the risk of insertional mutagenesis, the difficulty of large-scale production at Good Manufacturing Practice grades, limited cargo size, and cost-safety issues inherent to the biological properties.

[0161] The term "protein-based complex" refers to a compound formed by electrostatic interactions, or a combination of hydrophobic and electrostatic interactions, between an anionic polymer according to the present invention and a protein that is positively charged at a given pH, and which acts as a shielding layer on top of or intercalates between the protein.

[0162] According to a more specific embodiment, the N / P ratio in the polyplex of the present disclosure, defined as [total number of cationic groups in the block copolymer (N)] / [total number of phosphate groups in the nucleic acid (P)], is in the range of 1 to 200, preferably 2 to 100, and more preferably 2 to 50. The N / P ratio refers to the ratio of the molar concentration (N) of the protic amino groups derived from the side chains of the compound of formula (I) to the molar concentration (P) of the phosphate groups derived from the nucleic acid in the mixed solution.

[0163] According to a more specific embodiment, the + / - ratio between the cationic polymer or positively charged protein of the present disclosure and at least one shielding polymer is defined as [total number of positive charges originating from cationic groups in the block copolymer (+)] / [total number of negative charges originating from polyanionic blocks in the shielding polymer(s) (-)], which is in the range of 0 to 1, preferably 0.1 to 1, more preferably 0.3 to 1. The + / - ratio refers to the ratio of the molar concentration of the positive charges (+) originating from the protic amino groups originating from the side chains of the cationic polymer or positively charged protein in the mixed solution to the molar concentration of the negative charges (-) originating from the anionic blocks.

[0164] In more specific embodiments, the polymer complex may contain an amount of at least one active agent in the range of 1-50% w / w, based on the mass ratio of active agent to polymer complex. In preferred embodiments, the range is 1-30% w / w. In even more preferred embodiments, the polymer complex contains an amount of active agent in the range of 2-20% w / w. Other preferred ranges are 2-20% w / w, 3-15% w / w, and 3-7% w / w.

[0165] In more specific embodiments, the protein-based complex may contain an amount of protein in the range of 5-99% w / w, based on the mass ratio of active agent to protein-based complex. In preferred embodiments, the range is 15-98% w / w. In even more preferred embodiments, the protein-based complex contains an amount of protein in the range of 30-95% w / w. Other preferred ranges are 40-92% w / w, 50-90% w / w, and 60-90% w / w.

[0166] The pharmaceutical, diagnostic or theranostic compositions according to the present disclosure may be prepared in solid form or in aqueous suspension in a pharma- ceutically acceptable diluent. These preparations may be administered by any suitable route of administration, and thus the preparations are formulated in pharmaceutical forms suitable for the selected route of administration. In more particular embodiments, administration is by oral, topical, rectal or parenteral route (subcutaneous, intraperitoneal, intradermal, intramuscular, intravenous, etc.).

[0167] In view of the nomenclature used herein for the shielding polymers according to the invention, it should be noted that the numerical values ​​stated in brackets refer to the degree of polymerization (DP) of each monomer unit as a statistical number. The DP of a particular monomer unit is calculated by dividing the molecular weight of the polymer by the molecular weight of the monomer unit. The DP values ​​are subject to reasonable uncertainties due to the ring-opening polymerization mechanism, which in the context of the present invention can be considered to be within the range of ±20%, preferably ±15%, more preferably ±10%, even more preferably ±5%, and particularly preferably ±2%. Thus, for example, compound V1 (pSar(72)-b-pGlu(ONa)(9) is described as having a pSarDP of 72, a pGlu(ONa)DP of 9; the DP numbers quoted here are subject to reasonable uncertainties within the above ranges.

[0168] All terms used herein in this application are to be understood in their ordinary meaning as known in the art unless otherwise specified. Other, more specific definitions of certain terms used in this application are set forth below and are intended to be applied uniformly throughout the specification and claims, unless a definition expressly set forth otherwise provides a broader definition.

[0169] Throughout the description and claims, the word "comprise" and variations of that word are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" also includes "consisting of". Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the detailed description or may be learned by the practice of the present invention. The following examples and drawings are provided by way of illustration and are not intended to limit the present invention. Reference signs placed in parentheses in connection with the drawings and in the claims are intended only to enhance the completeness of the claims and are not to be construed as limiting the scope of the claims. Furthermore, the present invention includes all possible combinations of the specific preferred embodiments described herein.

[0170] While only a few examples are disclosed herein, other alternatives, modifications, uses and / or equivalents are possible. Moreover, all possible combinations of the described embodiments are covered. Thus, the scope of the disclosure should not be limited by the specific embodiments, but should be determined only by a fair reading of the following claims.

[0171] Working Example Example 1: Synthesis of shielded block copolymer PSar-b-PGlu(ONa) Synthesis of shielded copolymer PSar-b-PGlu(Ona) by block polymerization: [ka]

[0172] 1.1A General procedure for polymerization of PSar-b-PGluOtBu: [ka] Sarcosine N-carboxyanhydride was added to a Schlenk tube equipped with a stir bar and stopper. After three cycles of vacuum / N2, the mixture was dissolved in anhydrous DMF. Then, the initiator (n-butylamine or iPropylamine) diluted in DMF (2 mL) was added to the reaction mixture, which was stirred at 10° C. for 16 h. Once the consumption of NCA was confirmed by IR, glutamic acid t-tert butyl ester NCA was added to the reaction mixture dissolved in anhydrous DMF. The mixture was stirred at 10° C. for 16 h. Upon completion, the reaction mixture became clear and complete conversion of the monomer could be detected by IR. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The block copolymer was isolated as a white solid. Yield: 70-90%. The polymerization product was not characterized by NMR since it was only soluble in the deprotection solvent.

[0173] 1.2A General procedure for the deprotection step: [ka] The block copolymer of PSar-b-PGluOtBu was dissolved in trifluoroacetic acid (100 mg / mL) at 0° C., and the mixture was stirred at 5° C. for 1 h. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The block copolymer was isolated as a white solid. Yield: 95%. [V1-V2]1H NMR (D2O): δ 1.19 (d, J = 7.2 Hz,CH3), 1.90-2.53 (m, CH2),2.83-3.28 (m, CH3), 3.95-4.63 (m, CH2 PSar + CH GluOtBu). [V3]1H NMR (D2O): δ 1.19 (t, J = 7.2 Hz,CH3), 1.35 (m, CH2), 1.70-2.53 (m, CH2), 2.82-3.32 (m, CH3), 4.02-4.60 (m, CH2 PSar + CH GluOtBu). [Table 1]

[0174] Example 2. Synthesis of the shielded copolymer PSar-b-PGlu(Ona) by peptide coupling of two homopolymers: [ka]

[0175] 2.1 General procedure for ring-opening polymerization of MeA-PSar-Succ and nBu-PGluOBzl [ka] Sarcosine NCA was added to a Schlenk tube equipped with a stir bar and stopper. After three cycles of vacuum / N2, the mixture was dissolved in anhydrous DMF. Then, initiator (2-methoxyethylamine) diluted in DMF (2 mL) was added to the reaction mixture, which was stirred at 10° C. for 16 h. Once the consumption of NCA was confirmed by IR, powdered succinic anhydride (10 eq.) was added to the reaction mixture and stirred at room temperature for 16 h. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The homopolymer was isolated as a white solid. Yield: 70~90%.1H NMR (300 MHz, TFA): δ 2.71 (brs, 2H, CH2), 2.86-3.22 (m, 3H, CH3), 3.40 (s, 3H, CH3), 4.10-5.58 (m, 2H, CH2). [ka]

[0176] Benzyl-L-glutamate NCA was added to a Schlenk tube equipped with a stir bar and stopper. After three cycles of vacuum / N2, the mixture was dissolved in anhydrous DMF. Then, the initiator (n-butylamine) diluted in DMF (2 mL) was added to the reaction mixture and stirred at 10° C. for 16 h. Once the NCA was consumed, it was confirmed by IR. Powdered succinic anhydride (10 eq.) was added to the reaction mixture and stirred at room temperature for 16 h. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The homopolymer was isolated as a white solid. Yield: 70-90%. 1H NMR (300 MHz, TFA): δ 0.87 (t, J= 7.3 Hz, CH3);1.29 (dd, J= 7.2, 15.0 Hz, CH2), 1.46 (d, J= 8.0 Hz, CH2), 1.80-2.73 (m, CH2), 4.67 (m, CH), 5.00-5.24 (m, benzyl CH), 7.15-7.37 (brs, aryl CH): [Table 2]

[0177] 2.2 General procedure for peptide coupling reaction to produce MeA-PSar-succ-PGluOBzl-nBu copolymers: [ka] MeA-PSar-succ (0.32 mmol) was added to a two-necked round-bottom flask equipped with a stir bar and stopper, then purged with three cycles of vacuum / N2, and dissolved in 25 mL of DMF. CDI (2.1 equiv., 0.68 mmol) was then added to the reaction mixture and stirred at room temperature for 30 min. After this time, nBu-PBG (1.2 equiv., 0.38 mmol) dissolved in 8 mL of DMF was added. The mixture was stirred at room temperature for 16 h. The reaction mixture was poured into acetone to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The final product was isolated as a white solid. Yield: 60-70. 1H NMR (DMSO): δ 1.06 (t, J = 7.3 Hz, CH3), 1.48 (dd, J= 7.4, 14.8 Hz, CH2), 1.65 (dd, J= 7.1, 14.4 Hz, CH2), 2.01-2.83 (m, CH2), 3.17-3.55 (m, CH3), 3.72 (s, CH3), 3.81-4.06 (m, CH2), 4.44-4.94 (m, CH) 5.19-5.43 (m, benzyl CH), 7.24-7.61 (m, aryl CH). [Table 3]

[0178] 2.3 General procedure for the deprotection step: [ka] The block copolymer of MeA-PSar-succ-PBG-nBu was dissolved in THF (200 mg / mL). Aqueous NaOH was added to the mixture and stirred at 4° C. for 16 h. The reaction mixture was neutralized to pH 4 with 6 M HCl and poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The block copolymer was isolated as a white solid. Yield: 70~80%. [V_1]1H NMR (D2O): δ 0.91 (t, J= 7.0 Hz, CH3), 1.33 (d, J = 6.8 Hz,CH2), 1.50 (d, J = 6.8 Hz,CH2), 2.39 (m, CH2), 2.72-2.81 (m;CH2), 2.88-3.25 (m, CH3), 3.39 (s, CH3), 3.46 (m, CH2), 3.56 (m, CH2), 4.03-4.63 (m, CH2PSar + CH GluOtBu). [Table 4]

[0179] Example 3. Synthesis of shielded block copolymer Rn-PSar-b-PGlu(ONa) using a directing agent. Shielded block copolymers of Rn-PSar-b-PGlu(ONa) are synthesized using small organic molecules as initiators for polymerization. These specific organic motifs (TLR7, galactosamine and manosamine) could be used to target specific organs.

[0180] 3.1 Synthetic route for the shielded block copolymer Rn-PSar-b-PGlu(ONa) using a directing agent. 3.1.1 General procedure for the polymerization of directing agent (Rn)-PSar. [ka] *Note that mannosamine and galactosamine are shown in their linear forms in equilibrium with their multiple cyclic forms.

[0181] Sarcosine N-carboxyanhydride was added to a Schlenk tube equipped with a stir bar and stopper. After three cycles of vacuum / N2, the mixture was dissolved in anhydrous DMSO. Then, initiator (TLR7, galactosamine or mannosamine) diluted in DMSO (2 mL) was added to the reaction mixture and stirred at room temperature for 16 h. Once the consumption of NCA was confirmed by IR, the reaction mixture was poured into diethyl ether:THF (8:2) to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. Polysarcosine was isolated as a white solid. Yield: 70%. [X1-X2-

[0182] The presence of sugars is determined by the Benedict test, which is a chemical test that can be used to detect the presence of reducing sugars in a given analyte. Thus, simple carbohydrates that contain free ketone or aldehyde functions can be identified.

[0183] The Benedict test is performed by heating a reducing sugar with Benedict's reagent (a blue solution prepared by mixing copper sulfate pentahydrate, sodium citrate, and sodium carbonate in distilled water). If the color of the solution changes from blue to red, it indicates the presence of sugar in the polymer. [Table 5]

[0184] 3.2 General synthetic procedure for block copolymer directing agent-Rn-PSar-b-PGluONa. [ka] Glutamic acid t-tert-butyl ester NCA was added to a Schlenk tube equipped with a stir bar and stopper. After three cycles of vacuum / N2, the mixture was dissolved in anhydrous DMF. P-sarcosine (synthesized in the previous section) diluted in DMF (4 mL) was then added to the reaction mixture and stirred at 10° C. for 16 h. Once the consumption of NCA was confirmed by IR, the reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The block copolymer was isolated as a white solid. Yield: 80%. [5.1-5.2-5.3-5.4] 1H-NMR (TFA): δ 2.29-2.56 (s, CH3), 2.86-3.69 (m, CH2), 3.77-4.33 (m, CH3), 4.97 (brs, CH2), 5.22-5.78 (m, CH2PSar + CH GluOtBu). [Table 6]

[0185] 3.3 General procedure for the deprotection step: Deprotection was carried out according to the procedure described above in Example 1 to produce the corresponding shielded block copolymer with directing agent Rn-PSar-b-PGlu(ONa). Yield: 90%.

[0186] [A1-A2-A3-A4] 1H-NMR (D2O):1.67-2.46 (m, CH2);2.74-3.23 (m, CH3);3.71 (brs, CH2), 3.94 (s, CH2), 4.01-4.56 (m, CH2PSar+ CH PGA). [ka] [Table 7]

[0187] Example 4. Synthesis of shielded block copolymer PGlu-Diol-b-PGlu(ONa) 4.1 General procedure for polymerization of PGluOtBu: [ka] Glutamic acid t-tert-butyl ester NCA was added to a Schlenk tube equipped with a stir bar and stopper. After 3 cycles of vacuum / N2, the mixture was dissolved in anhydrous DMF. Then, initiator (n-butylamine) diluted in DMF (2 mL) was added to the reaction mixture and stirred at 10°C for 16 h. Once the consumption of NCA was confirmed by IR, the reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The homopolymer was isolated as a white solid. Yield: 70-90%.

[0188] The polymerization product is used without further characterization or purification, which is performed as it is deprotected.

[0189] 4.2 General procedure for the deprotection step: [ka]

[0190] Deprotection was carried out according to the procedure described above in Example 1 to yield the corresponding nBu-PGA. Yield: 90%.1H NMR (D2O): δ 0.85 (t, J = 7.03 Hz, CH3),1.28 (m, CH2),1.44 (m, CH2), 1.84-2.39 (m, CH2), 4.15 -4.46 (m, CH ).

[0191] 4.3 General procedure for the synthesis of diblock copolymer nBu-PGA- b -PGluOtBu. [ka]

[0192] The polymerization reaction was carried out according to the procedure described above in Example 3.1, in this case using nBuPGA as initiator. The block copolymer was isolated as a white solid. Yield: 70-80%. 1H NMR (TFA): δ 1.62 (t, J = 7.59 Hz), 1.99 (m, CH2), 2.09 (m, CH2), 2.12-2.40 (m, CH3), 2.72-3.59 (m, CH2), 5.49 (brs, CH).

[0193] 4.4 General procedure for peptide coupling with CDI for the synthesis of PGlu-diol-b-PGluOtBu: [ka]

[0194] nBuPGA-b-PGluOtBu (3.09 mmol) was added to a two-necked round-bottom flask equipped with a stir bar and stopper, then purged with three cycles of vacuum / N2, and dissolved in 6 mL of DMF. CDI (1.6 equiv., 4.95 mmol) was then added to the reaction mixture and stirred at room temperature for 30 min. After this time, 3-aminopropane-1,2-diol dissolved in 2 mL of DMF was added. The mixture was stirred at room temperature for 16 h. The reaction mixture was poured into acetone to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The final product was isolated as a white solid. Yield: 60-70.

[0195] 1H NMR (DMSO): δ 0.85 (t, J = 7.2 Hz, CH3), 1.20-1.48 (m, CH3), 1.65-2.31 (m, CH2), 2.98 (brs, CH), 4.16 (brs, CH), 4.52 (brs, CH), 4.72 (brs, CH), 7.74 (brs, NH-amide), 8.10 (brs, NH-amide). [Table 8]

[0196] 4.5 General procedure for the deprotection step: [ka] Deprotection was carried out according to the procedure described above in Example 1 to produce the corresponding block copolymer PGlu-diol-b-PGlu(ONa). Yield: 90%. 1H NMR (D2O): δ 0.96 (t, J = 7.12 Hz, CH3), 1.36 (dd, J = 15.0, 7.2 Hz, CH2);1.54 (d, J =7,12 Hz), 1.88-2.80 (m, CH2), 3.18-3.48 (m, CH2);3.52-3.74 (m, CH2), 3.83 (brs, CH), 4.37 (brs, CH): [Table 9]

[0197] Example 5. Synthesis of shielded block copolymer PSar-R5(W) 5.1 Procedure for polymerization of Boc-NH-ethyl-R5 random copolymers. [ka] Glutamic acid t-tert-butyl ester NCA, leucine NCA and alanine NCA were added to a Schlenk tube equipped with a stir bar and stopper. After three cycles of vacuum / N2, the mixture was dissolved in anhydrous mixture of DMF:THF (1:1). Then, initiator (N-Boc-ethylenediamine) diluted in DMF (2 mL) was added to the reaction mixture, which was stirred at 10°C for 16 hours. Once the consumption of NCA was confirmed by IR, the reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The random copolymer was isolated as a white solid. Yield: 70~80%. 1H NMR (300 MHz, TFA): δ 1.51 (d, J = 8.9 Hz, CH3), 1.94-2.22 (m, CH2), 2.80 (m, CH2), 3.142 (m, CH3), 4.05 (brs, CH2), 4.31 (brs, CH2), 4.88-5.44 (m, CH).

[0198] 5.2 General procedure for one-pot polymerization / capping of Boc-NH-ethyl-R5-b-PSar: [ka]

[0199] Sarcosine N-carboxyanhydride was added to a Schlenk tube equipped with a stir bar and a stopper. After three cycles of vacuum / N2, the mixture was dissolved in anhydrous DMF. Then, the initiator (Boc-NH-ethyl-R5 random copolymer) diluted in DMF (2 mL) was added to the reaction mixture, which was stirred at 10° C. for 16 h. Once the consumption of NCA was confirmed by IR, acetic anhydride (10 eq., 1.47 mmol) and DIPEA (1 eq., 0.147 mmol) were added to the reaction mixture. The mixture was then stirred at 10° C. for 2 h. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. Yield: 60~70%. 1H NMR (300 MHz, TFA): δ 1.69 (d, J = 9.0 Hz, CH3), 2.10-2.47 (m, CH2), 2.97 (m, CH2), 3.25 (s, CH3), 3.40 (brs, CH2), 3.75-4.09 (m, CH3), 5.05-5.41 (m, CH + CH2). [Table 10]

[0200] 5.3 General procedure for the deprotection step: [ka] The block copolymer of Boc-NH-ethyl-R5-b-PSar was dissolved in trifluoroacetic acid (100 mg / mL) at 0° C., and the mixture was stirred at 5° C. for 2 h. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The isolated polymer was neutralized to pH 6.5-7 and purified by centrifugal ultrafiltration. After filtration, the remaining aqueous polymer solution was freeze-dried to obtain the final product. Yield:70~80%(W1)(D2O): δ 0.98 (d, J = 14.2 Hz, CH3), 1.62 (m, CH3 Ala + CH Leu), 1.96-2.62 (m, CH2), 2.77-3.38 (m, CH3), 3.63 (brs, CH2), 4.01 (brs, CH2), 4.05-4.64 (m, CH3 Sar + CH Glu, Ala, Leu). [Table 11]

[0201] Example 6. Synthesis of shielded detachable block copolymer PSar-succ-detachable-GALA-like random copolymer (Y) 6.1 General procedure for polymerization and endcapping of PSar-Succ. [ka]

[0202] The experimental procedure is the same as that described in section 1.1B.

[0203] 6.2 General procedure for masked cleavable MeA-PSar-succ cysteamine-Fmoc. [ka] PSar-Succ (0.18 mmol, 1 g) was added to a two-necked round-bottom flask equipped with a stir bar and stopper, purged with three cycles of vacuum / N2, and dissolved in 4 mL of DMF. CDI (2.1 eq, 0.38 mmol) was then added to the reaction mixture and stirred at room temperature for 30 min. After this, Fmoc-cysteamine (1.2 eq, 0.21 mmol) and DIPEA (1 eq, 0.21 mmol) dissolved in 30 mL of DMF were added. The mixture was stirred at room temperature for 16 h. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The final product was isolated as a white solid. These systems are analyzed after the fmoc deprotection reaction.

[0204] 6.3 General procedure for Fmoc deprotection: [ka] MeA-PSar-Succ-Cysteamine-Fmoc (1 g) was added to a two-necked round-bottom flask equipped with a stir bar and stopper, purged with three cycles of vacuum / N2, and dissolved in 10 mL of DMF. Piperidine (2 mL) was then added to the reaction mixture and stirred at room temperature for 2 h. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The final product was isolated as a white solid. Yield: 95%.1H NMR (300 MHz, TFA): δ 2.50 (m, CH2), 2.74 (m, CH2), 2.84-3.24 (m, CH2), 3.38 (s, CH3), 3.54 (m, CH2), 4.28 (m, CH2).

[0205] 6.4 Copolymer PSar-succ-detachable-GALA-like random copolymer (Y) polymerization procedure. [ka] Glutamic acid t-tert-butyl ester NCA, leucine NCA and alanine NCA were added to a Schlenk tube equipped with a stir bar and stopper. After three cycles of vacuum / N2, the mixture was dissolved in anhydrous mixture of DMF:THF (1:1). Then, initiator (MeA-PSar-Succ-Cysteamine) diluted in DMF (2 mL) was added to the reaction mixture and stirred at 10°C for 16 hours. Once the consumption of NCA was confirmed by IR, the reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The random copolymer was isolated as a white solid. Yield: 60~70%. 1H NMR (300 MHz, TFA): δ 1.07 (d, J = 8.9 Hz, CH3), 1.63 (brs, CH3), 1.69-2.32 (m, CH2), 2.78 (m, CH2), 2.96-3.56 (m, CH3), 3.70 (s, CH3), 3.84 (M, CH2), 3.96 (M, CH2), 4.42-5.06 (m, CH2). [Table 12]

[0206] 6.5 General procedure for the deprotection step: [ka] The block copolymer of PSar(50)-succ-detachable-[PGlu(OtBu)(6)-co-PAla(10)-co-PLeu(5)] was dissolved in trifluoroacetic acid (100 mg / mL) at 0 °C, and the mixture was stirred at 5 °C for 2 h. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The isolated polymer was neutralized to pH 6.5-7 and purified by centrifugal ultrafiltration. After filtration, the remaining aqueous polymer solution was lyophilized to obtain the final product. Yield: 70~80% (Y1) 1H NMR (300 MHz (D2O): δ 0.98 (d, J = 20.6 Hz, CH3), 1.53 (brs, CH3 Ala + CH Leu), 1.65-2.44 (m, CH2), 2.69 (m, CH2), 3.60 (s, CH3), 3.75 (m, CH2), 3.86 (m, CH2), 4.05-4.64 (m, CH3Sar + CH Glu, Ala, Leu). [Table 13]

[0207] Example 7. Synthesis of a comparative example in which (R5) contains only one of the disclosed groups according to the present invention: PGluONa-co-PLeu-b-PSar copolymer (E) The following examples were synthesized for comparison purposes according to the procedure described below: Tian, ​​C., Ling, J., & Shen, Y. qing. (2015). Self-assembly and pH-responsive properties of poly(L-glutamic acid-rL-leucine) and poly(L-glutamic acid-rL-leucine)-b-polysarcosine. Chinese Journal of Polymer Science (English Edition), 33 (8), 1186-1195. https: / / doi.org / 10.1007 / s10118-015-1669-0. The following products were further used to stabilize different polyplexes formed by either N1 or PEI, with negative results (see Examples 10.6 and 10.9).

[0208] 7.1 General procedure for polymerization of PSar-b-PGluOtBu [ka] Glutamic acid t-tert-butyl ester NCA and L-leucine NCA were added to a Schlenk tube equipped with a stir bar and stopper. After three cycles of vacuum / N2, the mixture was dissolved in anhydrous DMF (150 mg / mL). The initiator (benzylamine) diluted in DMF (2 mL) was then added to the reaction mixture and stirred at 10°C for 16 hours. Once the consumption of NCA was confirmed by IR, sarcosine-N-carboxyanhydride (Sar-NCA) was added to the reaction mixture dissolved in anhydrous DMF. The mixture was stirred at 10°C for 16 hours. Upon completion, the reaction mixture became clear and complete conversion of the monomer could be detected by IR. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The block copolymer was isolated as a white solid. Yield: 70-90%. The polymerization product was not characterized by NMR since it was only soluble in the deprotection solvent.

[0209] 7.2 General procedure for the deprotection step: [ka] The block copolymer of Bn-PSar-b-PGluOtBu was dissolved in trifluoroacetic acid (100 mg / mL) at 0° C., and the mixture was stirred at 5° C. for 1 h. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The block copolymer was isolated as a white solid. Yield: 95%. [V1-V2]1H NMR (D2O): δ 0.93 (d, J = 27.0 Hz,CH3), 1.62 (brs, CH2), 1.73 (brs, CH), 1.82-2.45 (m, CH2), 2.78-3.17 (m, CH3), 4.00-4.63 (m, CH2PSar + CH GluONa), 7.38 (m, aryl CH). [Table 14]

[0210] Example 8. Synthesis and description of polycationic carriers To demonstrate the stabilization capabilities and enhanced transfection functions conferred to polyplexes by the shielding polymer, the universal benchmark jetPEI was used (Polyplus-transfection S.A, Illkirch, France) (Ref Polyplus: 101-10N). JetPEI® is a powerful reagent that ensures robust, efficient and reproducible DNA transfection in mammalian cells with low toxicity. JetPEI® is mainly composed of linear polyethylenimine produced by Polyplus-transfection. JetPEI® is provided as a 7.5 mM solution in sterile non-pyrogenic water (expressed as the concentration of nitrogen residues). Also, to demonstrate the stabilization of structurally and compositionally very different alternative polycations, different polycationic NVVs based on star-shaped polyamino acids were synthesized and their polyplexes were also stabilized and assayed. The following examples describe the preparation of the aforementioned polycationic compounds used for complexation and vehiculization of genetic material.

[0211] 8.1 Preparation of compound N1 [ka]

[0212] Generally speaking, to synthesize the compound of formula (N1) according to the present disclosure, a three-arm star initiator was first obtained within 2-3 steps. Such initiator was then used to polymerize γ-benzyl L-aspartate-NCA and L-phenylalanine-NCA to produce benzyl-protected star-shaped random copolymer (St-PAsp(Bz)-co-PPhe). The benzyl group was removed by aminolysis reaction to obtain the corresponding Star-PAsp-oligoamine-co-PPhe.

[0213] Scheme 1 shows a specific example of the polymerization and aminolysis steps: [ka]

[0214] 8.1. Synthesis of A-arm star initiators The synthetic route to the three-arm star initiator is described below. Example 8.1.A: Trifluoroacetate salt of N,N,N-tris(2-((2-aminoethyl)disulfanyl)ethyl)benzene-1,3,5-tricarboxamide (St-SS-initiator) (15)

[0215] The trifluoroacetate salt of N,N,N-tris(2-((2-aminoethyl)disulfanyl)ethyl)benzene-1,3,5-tricarboxamide (St-SS-initiator) (17) was synthesized according to the general procedure of Scheme 2 disclosed herein. [ka]

[0216] The synthesis of the trimeric amine initiator begins with a coupling reaction followed by deprotection of the amine.

[0217] Step (a) Synthesis of tri-tert-butyl ((((benzenetricarbonyltris-(azanediyl))tris(ethane-2,1-diyl))tris(disulfanediyl))tris(ethane-2,1-diyl))-tricarbamate: [ka]

[0218] N-(tert-butyloxycarbonyl)cystamine (7.99, 27 mmol, 3.3 equiv.) was weighed into a flame-dried two-necked round-bottom flask and dissolved in 56 mL of anhydrous THF. Freshly distilled DIPEA (4.75 mL, 27 mmol, 3.3 equiv.) was added and stirred at room temperature for 15 min. 1,3,5-benzenetricarbonyl trichloride (2.25 g, 8.3 mmol, 1 equiv.) was weighed into a flame-dried two-necked round-bottom flask and dissolved in 28 mL of anhydrous THF. The trichloride solution was slowly added to the N-(tert-butyloxycarbonyl)cystamine mixture via syringe. The progress of the reaction was monitored by thin layer chromatography (TLC). After 4 h, the solvent was evaporated in vacuum and the residue was dissolved in ethyl acetate. The organic layer was washed successively with Milli-Q water, 1 M hydrochloric acid and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate and concentrated in vacuo to give tri-tert-butyl ((((benzenetricarbonyltris-(azanediyl))tris(ethane-2,1-diyl))tris(disulfanediyl))tris(ethane-2,1-diyl))-tricarbamate as a white foam (7.5 g, η=98%). 1 H NMR (CDCl3): δ = 1.39 (brs, 27H, -C(CH3)3), 2.84 (t, J = 6.26 Hz, 6H, CH2), 2.96 (t, J = 6.84 Hz, 6H.CH2), 3.46 (m, 6H, CH2), 3.79 (m, 6H, CH2), 5.18 (brs, 3H, -NHBoc), 7.39 (brs, 3H, aryl CH).

[0219] Step (b): Synthesis of the trifluoroacetate salt of N,N,N-tris(2-((2-aminoethyl)disulfanyl)ethyl)benzene-1,3,5-tricarboxamide (St-SS-initiator) (15): [ka]

[0220] 7.5 g (8.19 mmol) of initiator (15) was dissolved in anhydrous dichloromethane (180 mL) and 90 mL of TFA was added. The reaction was stirred under nitrogen for 60 min and completion of the reaction was monitored by TLC. The solvent was evaporated under vacuum. The TFA salt of initiator (15) (7 g, 7.31 mmol) was obtained in quantitative yield and dried under vacuum. 1H NMR (D2O): δ = 2.86 (m, 12 H), 3.25 (t, J = 6.49 Hz, 8H), 3.60 (t, J = 6.85 Hz, 8H), 8.02 (brs, 3H, aryl CH).

[0221] Example 8.1.B. Synthesis of Star-PAsp(Bz)(16) copolymers containing hydrophobic fragments. [ka] To synthesize copolymers bearing hydrophobic residues, polymerization was carried out via a ring-opening polymerization mechanism using the trifluoroacetate salt of N,N,N-tris(2-((2-aminoethyl)disulfanyl)ethyl)-benzene-1,3,5-tricarboxamide as the initiator.

[0222] General procedure for the synthesis of St-SS-PAsp(Bz)(45)-co-PLeu (5): β-Benzyl-L-aspartic acid-N-carboxyanhydride (3.5 g, 14.15 mmol) and L-phenylalanine-N-carboxyanhydride (1.57 mmol) were added to a Schlenk tube equipped with a stir bar and stopper, purged with three cycles of vacuum / N2, and dissolved in a mixture of anhydrous chloroform (100 mL) and DMF (6 mL). The star-shaped initiator was then dissolved in DMF (4 mL) and added to the reaction mixture. The mixture was stirred at 50° C. for 16 h. Upon completion, the reaction mixture became clear and complete conversion of the monomers could be detected by IR. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The copolymer was isolated as a white solid. Yield: 70-80%. 1H NMR (TFA): δ = 2.99 (s, 2H, CH), 3.94 (brs, 1H, CH), 4.93 (s, 1H, CH), 5.15 (m, 2H, benzyl CH2), 7.20 (s, 5H, aryl CH), 8.42 (s, aryl CH).

[0223] The rate of introduction of repeat units was controlled by varying the mixture ratio of the corresponding monomer units reacted. In this precursor, the hydrophobic residues match the protecting groups of polyaspartic acid by 1H-NMR. These systems are analyzed after aminolysis reaction (Example 5.1.C below).

[0224] Example 8.1.C Synthesis of amphiphilic polyaspartamide derivative St-SS-PAspDET-co-PR18(X). Polyamino acids were prepared by simultaneous aminolysis of PBLA and DET, as shown in the synthetic route (7) below. [ka]

[0225] As an example, a synthesis method is described herein in which R18 represents a phenylalanine group. Copolymer of St-SS PAsp(Bz)45-co-PPhe (5) (500 mg copolymer, 470 mg PBLA, DP: 45) was dissolved in NMP (10 mL) and cooled to 4°C. The resulting copolymer solution was added dropwise to a mixture of DET (12 mL, 50 equivalents relative to the PAsp(Bz) unit) and the solution was stirred for 4 hours at 4°C under nitrogen atmosphere. After this time, the reaction mixture was added dropwise into cold HCl 6M for neutralization (pH 3.5). The polymer product was purified by centrifugal ultrafiltration. After filtration, the remaining aqueous polymer solution was lyophilized to obtain the final product. Yield: 70-80%. 1H NMR (DO) [R18 = Phe side chain]: δ = 2.91 (brs, 2H, CH2), 3.84-3.18 (m, 2H, CH2), 7.34 (brs, 5H, aryl CH of Phe), 8.33 (s, aryl CH).

[0226] Table 15 refers to the amphiphilic copolymer St-SS-PAspDET-co-R18 according to formula (N1). [Table 15]

[0227] 8.2 Preparation of compound N2 [ka] Example 8.2.A: St-Poly(β-benzyl-L-aspartic acid) (Star-PAsp(Bz)) (Pn)

[0228] The general procedure for the polymerization of St-PAsp(Bz) (6) is as follows: [ka]

[0229] The synthesis of compound N2 is very similar to that described in the previous example, beginning with the same initiator as previously described. β-benzyl-L-aspartate-NCA (5 g, 2 mmol) was added to a Schlenk tube equipped with a stir bar and stopper, purged with three cycles of vacuum / N2, and dissolved in a mixture of anhydrous chloroform (100 mL) and DMF (6 mL). Next, star-shaped initiator (St) was dissolved in DMF (4 mL) and added to the reaction mixture. The mixture was stirred at 50° C. for 16 h. Upon completion, the reaction mixture became clear and complete conversion of the monomer could be detected by IR. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. St-poly(β-benzyl-L-aspartate) (Star-PAsp(Bz)) (Pn) was isolated as a white solid. Yield: 70-90%. 1 H NMR (TFA): δ = 2.92 (m, 2H, CH2), 4.85 (s, 1H, CH), 5.05 (m, 2H, benzylCH2), 7.13 (s, 5H, arylCH), 8.38 (s, arylCH). [Table 16]

[0230] Example 8.2.B Aminolysis of Poly(β-benzyl-L-aspartate) (6) to Obtain St-PAsp-DET(Nn) General procedure for the aminolysis reaction to produce the polycationic homopolymer PaspDET regardless of the nature of the initiator used in the polymerization step: [ka]

[0231] St-PAsp(Bz) (6) (DP=50, 750 mg) was dissolved in NMP (15 mL) and cooled to 4 °C. This solution was added dropwise to DET (50 equivalents of DET per Asp unit, 19 mL) cooled at 4 °C, and the mixture was stirred at the same temperature for 4 h. After this time, the reaction mixture was added dropwise to cold HCl 6 M for neutralization (pH 3.5). The polymer product was purified by centrifugal ultrafiltration. After filtration through a 0.22 μm PES filter, the remaining aqueous polymer solution was lyophilized to give the final product (370 mg, η=50%). 1 H NMR (D2O): δ 2.93 (brs, 2H, CH2), 3.12-3.85 (m, 2H, CH2), 8.33 (s, 3H, arylCH). [Table 17]

[0232] Example 9. Polyplex Formulations Polyplex formulations are referred to as "PX n_ratio1_Shielding polymer_ratio2 nuc " where "n" corresponds to the nomenclature of the polycationic compounds used to form the polyplexes given above; where "ratio1" refers to the N / P ratio of cationic polymer to genetic material, "shielding polymer" refers to the polyanionic shielding diblock copolymer, "ratio2" refers to the + / - ratio of cationic polymer to shielding (anionic) polymer, and "nuc" refers to the type of nucleic acid: pDNA, clDNA or mRNA.

[0233] In the following examples, pDNA (purchased from PlasmidFactory, reference PF461 (pCMV-luc), containing 6233 bp expressing luciferase) and clDNA according to SEQ ID NO: 1 obtained according to standard molecular biology methods (for example those disclosed in Heinrich, M. et al. "Linear closed mini DNA generated by the prokaryotic cleaving-joining enzyme TelN is functional in mammalian cells", J Mol Med, 2002, vol. 80, pp. 648-654) were used. mRNA was purchased from Trilink as reference L-1201-1000 CleanCap Fluc mRNA (5 moU) expressing luciferase as a reporter gene.

[0234] The sequence of the cIDNA according to SEQ ID NO: 1 in the examples is the sequence of Table 18. [Table 18] TIFF2024525935000061.tif198159TIFF2024525935000062.tif193159TIFF2024525935 000063.tif198159TIFF2024525935000064.tif195159TIFF2024525935000065.tif90159

[0235] 9.1. Polyplex Formulation Procedure 1 Shielded polyplex formulations for testing stability, size, toxicity, and transfection ability were prepared in-situ (pipette mixing) as follows:

[0236] The desired amount of pDNA, clDNA or mRNA and the calculated amount of cationic polymer with the indicated charge ratio (+ / -) or amine to phosphate ratio (N / P) were diluted in PBS at pH 7.4 in separate tubes. Only protonatable nitrogens, not amide nitrogens, were considered in the calculation of + / - and N / P ratios. Prior to polyplex formation, the corresponding amount of shielding polymer was added to the nucleic acid tube and mixed. For the formation of shielded polyplexes, the cationic polymer solution and the genetic material + shielding polymer solution were mixed by rapid pipetting up and down (10 times) and incubated for 20 min at room temperature. The formed polyplexes were then characterized by DLS to determine the size and Z potential.

[0237] As a specific example, shielded polyplex PX loaded with 20 μg of pDNA (final volume of polyplex 200 μl) N1_8_V1_1 pDNA The formulation of other polyplexes is carried out in a similar manner. The amount of shielded anionic polymer according to the present invention is calculated as follows: once the amount of amines required for the NP8 ratio is established, divide them by 2, half of which is used for the polymer-DNA interaction and the other half is used for the required shielded anionic polymer NP ratio (NP1).

[0238] First, a 10 mg / ml stock solution of the shielding polymer in water and a 4 mg / ml stock solution of the polycationic polymer in water were prepared. The experimental procedure was carried out as follows: 1. Add 80 μl of PBS to an Eppendorf tube. Then dilute 20 μl of pDNA from a 1 mg / ml stock and add 15.8 μl of shielding polymer from a 10 mg / ml stock (final volume of 115.8 μl). 2. Add 18.2 μl of polycationic polymer solution (stock 4 mg / ml) to the pDNA-shielding polymer solution and bring to a final volume of 200 μl (66 μl PBS) with PBS. 3. Incubate at room temperature for 20 minutes. 4. Polyplexes are ready to use.

[0239] Samples formulated by this first method were prepared in a similar manner for in-vitro testing. After 24 hours of incubation, toxicity and transfection capacity are evaluated. The ratios tested for each polymer were N / P8, 15, or 30. As a positive control for transfection, jetPEI® (Polyplus-transfection S.A, Illkirch, France) (Ref Polyplus:101-10N) was used at a nitrogen to phosphorus ratio (NP5). Cell transfections were performed using jetPEI® according to the manufacturer's instructions. jetPEI® is mainly composed of linear polyethyleneimine produced by Polyplus-transfection. jetPEI® is provided as a 7.5 mM solution (expressed as the concentration of nitrogen residues) in sterile, non-pyrogenic water.

[0240] 9.2. Polyplex Formulation Procedure 2 A microfluidic device was used for this procedure of polyplex formulation. The microfluidic device was placed in a laminar flow hood to avoid possible contamination of the samples, and all polymers used in this formulation step were pre-sterilized by passing them through a 0.22 μm PES filter. For all microfluidic experiments, the microfluidic device was at room temperature.

[0241] As a specific example, shielded polyplex PX loaded with 3 μg of clDNA (final volume of polyplex 200 μl) N1_30_V1_1 clDNA The formulation of other polyplexes is carried out in a similar manner. First, a 50 mg / ml stock solution of shielding polymer and a 10 mg / ml stock solution of polycationic polymer were prepared. The experimental procedure was carried out as follows: 1. Add 75.81 μl of PBS to an Eppendorf tube. Then dilute 24.19 μl of clDNA from a 0.124 mg / ml stock and add 1.78 μl of shielding polymer from a 50 mg / ml stock (final volume of 101.78 μl). 2. Add 94.13 μl of PBS to another Eppendorf tube and dilute 4.09 μl from the polycationic polymer solution (stock 10 mg / ml) (final volume of 98.22 μl). 3. Both solutions were then loaded into 1 ml plastic syringes (BD Plastipak™, Spain). Two programmable injection syringe pumps (NE-4000, SyringePump, USA) were then used for fluid injection and control according to the desired flow rate ratio of the side and central streams (300 μl / min). 4. The final polyplex solution was collected and allowed to stabilize for 20 minutes. 5. Polyplexes are ready to use.

[0242] 9.2.A. Microfluidic Device and Setup The microfluidic device was purchased from Little Things Factory GmbH (Germany). The system consists of two connected reactors made of borosilicate glass: the first reactor (LTF-MS) has two inlet channels (one for DNA and the other for polymer) and one outlet channel, volume 0.2 ml, channel size: 1 mm, 0.5-20 ml / min / channel, not sensitive to blockages. Size: 115x60x6 mm (l, w, h). The second reactor (LTF-VS) has one inlet channel (connected to the outlet channel of the first reactor) and one outlet channel, volume 1.1 ml, channel size: 1 mm. Size: 115x60x6 mm (l, w, h). The first reactor is used for mixing and formation of polyplexes, the second reactor is used to extend the residence time.

[0243] In addition, two programmable pumps control the fluid flow rate of the syringes (NE-4000 Programmable Two-Channel Syringe Pump, Syringe Pump, USA). The system accepts injection rates from 1.436 μL / h (1 mL syringe) to 7515 mL / h (60 mL syringe).

[0244] This methodology provides reproducibility in polyplex formation as well as the potential for scale-up of the process.

[0245] Example 10 Size and stability of shielded polyplexes Polyplex stability is the most important aspect in developing efficient therapeutics. After confirming the mid- to long-term stability of the pharmaceutical formulation, a panel of assays will be performed that mimic the physiological conditions met by drugs according to the route of administration that require them to be stable during circulation to the target site of action. It is well known that polyplexes that exhibit a positive surface charge undergo salt-induced aggregation, which can cause inaccurate cell biological evaluations and serious toxicity issues when applied systemically. Initial stability studies are currently being developed during this project and they are aimed at monitoring the properties (size) of polyplex particles.

[0246] The size and Z potential of stabilized polyplexes formed with clDNA, mRNA, or pDNA at different N / P ratios, different polycations, and shielding polymers (V1 or V0.5) were performed using a Malvern Zetasizer NanoZS instrument equipped with a 532 nm laser at a fixed scattering angle of 173°. 20 μl samples were measured using a quartz glass high performance cuvette (Hellma Analytics). Size distribution was measured (diameter, nm) by measuring n>3. For stability measurements, polyplexes were kept in a refrigerator during the experiment, and the stability of polyplexes was measured at various times.

[0247] The cIDNA sequence shown in this example, SEQ ID NO:1, is the sequence in Table 18 above.

[0248] 10.1. Stabilization of polyplexes formed by N1 and V1 The stability and formation of N1 polyplexes at different times according to polyplex formulation procedure 1 (as reported in Example 8.1 above) was investigated using NP ratios (8 and 15) and shielding polymer V1 with different + / - charge ratios in PBS pH 7.4. Different amounts of genetic material were also used for this experiment (shown in Table 19). The final polyplex solutions (200 μl) were allowed to stabilize for 20 minutes before being sized by DLS (Malvern Panalytical, Spain). The polyplexes were kept in a refrigerator during the experiment and the stability of the polyplexes was measured at various times.

[0249] As shown in Table 19, the presence of the shielding polymer provided improved stability to the polyplexes in solution, maintaining a constant size over time and avoiding aggregation, up to at least several days. [Table 19]

[0250] As can be observed in the table, the size of the polyplexes depends on the mass of genetic material and the ratio of shielding polymer present in the final formulation. Those polyplexes formulated without a shielding polymer (i.e., PX N1_8_V1_0 pDNA and P.X. N1_8_V1_0 mRNA ) produced large aggregates that could not be measured by DLS techniques.

[0251] 10.2. Stabilization of polyplexes formed by N1 and V2 As shown in Table 20, the presence of the shielding polymer provided improved stability to the polyplexes formulated by the procedure set forth in Example 8.1 in solution for at least several days, maintaining a constant size over time and avoiding aggregation. [Table 20]

[0252] 10.3. Stabilization of polyplexes formed by N1 and W1 As shown in Table 21, the presence of the shielding polymer provided improved stability to the polyplexes formulated by the procedure set forth in Example 6.1 in solution for at least several days, maintaining a constant size over time and avoiding aggregation. [Table 21]

[0253] 10.4 Stabilization of polyplexes formed by N1 and Y1 As shown in Table 22, the presence of a shielding polymer provided stability to polyplexes formulated by the procedure set forth in Example 6.1 in solution. [Table 22]

[0254] 10.5 Stabilization of polyplexes formed by N1 and F1 As shown in Table 23, the presence of a shielding polymer provided stability to polyplexes formulated by the procedure set forth in Example 9.1. [Table 23]

[0255] 10.6 Stabilization of polyplexes formed by N1 and Comparative Example E1 As shown in Table 24, the presence of a shielding polymer did not confer stability to polyplexes formulated by the procedure set forth in Example 6.1 in solution. [Table 24]

[0256] 10.7 Change in polyplex Z potential upon addition of V1 shielding polymer to polyplexes formed with N1 Z-potential is an important feature of polyplexes to ensure shielding and avoidance of aggregation. Increasing amounts of shielding V1 were used to shield the same polyplexes formed with N1 and pDNA according to formulation protocol 1. Despite the stabilization of size and prevention of aggregation, a decrease in Z-potential can be clearly seen as the shielding polymer ratio increases. This decrease in Z-potential confirms that shielding of positive charges was efficiently achieved (Table 25). [Table 25]

[0257] 10.8. Stabilization of Polyplexes Formed by JetPEI Comparing Two Formulation Procedures The size and stability variations of stabilized polyplexes containing pDNA and formed with jetPEI (NP=5) and stabilized with different shielding polymer V1 + / - ratios and formulation procedures are evaluated by DLS. As shown in Table 26, the presence of the shielding polymer provided improved stability to the polyplexes in solution for up to 96 hours, maintaining a constant size over time and avoiding aggregation. [Table 26]

[0258] 10.9. Stabilization of polyplexes containing E1 and formed by jetPEI The variation in size and stability of stabilized polyplexes formed with jetPEI (NP=5) containing pDNA and stabilized with different + / - ratios of shielding polymer E1 and formulation procedure 1 (described in Section 8.1) was evaluated by DLS. As shown in Table 27, the presence of the shielding polymer did not confer stability to the polyplexes in solution. [Table 27]

[0259] 10.10. Stabilization of polyplexes formed by N2 and V1 The stability and formation of N2 polyplexes at different times from polyplex formulation procedure 1 (as reported above) was investigated using a NP ratio of 8 in PBS pH 7.4 and using different + / - charge ratios of shielding polymer V1. The presence of the shielding polymer provided improved stability to the polyplexes in solutions containing both pDNA and mRNA, maintaining a constant size over time and avoiding aggregation (Table 28). [Table 28]

[0260] 10.11. Stabilization of polyplexes formed by N1 and clDNA Stabilization of polyplexes formed by N1 and clDNA was performed using a microfluidic device. Polyplex stability and formation was performed in PBS pH 7.4 using an NP ratio of 30 for polyplex formation and an NP ratio of 1 for shielding polymer (V1). As shown in Table 29, the presence of the shielding polymer provided improved stability to polyplexes in solution.

[0261] The sequence of the cIDNA shown in this example, SEQ ID NO:1, is the sequence in Table 18 above. [Table 29]

[0262] Example 11. Complexation / decomposition experiments. Furthermore, the effectiveness of complexation and the possible presence of free pDNA in the polyplexes were evaluated using electrophoretic gels as a first screening method. To perform the electrophoresis, an E-gel Power Snap Electrophoresis device and an E-Gel Power Snap Camera (Invitrogen) were used. A 1.2% agarose gel prepared to contain SYBR safe DNA marker (E-Gel® 1.2% with SYBR safe, Invitrogen) was used. The complexation efficiency of polyplexes (20 μl) with different NPs and different + / - shielding polymer ratios was evaluated, as was the degradation of polyplexes in the presence of low (0.075 IU / ml) and high (200 IU / ml) heparin concentrations (PanReacAppliChem, Spain). For low concentrations, 0.1 μl of 15 IU / ml heparin solution was added to 20 μl of already formed polyplexes, and for high concentrations, 0.8 μl of 5000 IU / ml heparin solution was added to 20 μl of polyplexes. Once the gel is loaded (20 μl / well), the protocol of the instrument is selected depending on the type of gel used (in this case the protocol is about 40 minutes, but the time can be modified depending on the sample). [Table 30]

[0263] In all cases, no free pDNA is observed with the different NPs or at low concentrations of heparin, but at high concentrations of heparin, free pDNA signals are observed due to competition between heparin and pDNA binding to the polymer, indicating the ability of the polymers to release their cargo (representative images of the gels can be seen in Figure 1).

[0264] Example 12. In vitro biological testing Example 12A. Cell Culture HeLa cells were cultured in high glucose DMEM with Glutamax (Gibco-ThermoFisher #61965-059) supplemented with 10% fetal bovine serum (Hyclone #SV30160.03HI, provided by GE Healthcare Europe GmbH). Transfections were performed in 96-well plates with 10000 cells / well in a final volume of 100 μl and cells were incubated for 24 h at 37°C and 5% CO2. After 24 h, the medium was removed and refreshed with 90 μl of complete medium. Transfection mixtures were prepared using PBS and, in the case of the positive control (JetPEI), 10 μl of each formulation was added to the cells after 20 min stabilization according to the manufacturer's guidelines (#101-10N, Polyplus Transfection). After 24 h, cells were harvested and processed.

[0265] HEK293 (human embryonic kidney) cells were cultured in high glucose DMEM (Gibcoref 61965-059) + 10% FBS (Hyclone #SV30160.03HI, provided by GE Healthcare Europe GmbH). Transfections were performed in 96-well plates with 10000 cells / well in a final volume of 100 μl and cells were incubated for 24 h at 37°C and 5% CO2. After 24 h, the medium was removed and refreshed with 90 μl of complete medium. Transfection mixtures were prepared using PBS and, in the case of the positive control (JetPEI), following the manufacturer's guidelines (#101-10N, Polyplus Transfection), 10 μl of each formulation was added to the cells after 20 min stabilization. After 24 h, cells were harvested and processed.

[0266] Example 12B. ATP evaluation for cytotoxicity assessment After 24 hours of incubation, the medium was aspirated and 50 μl / well of ATPLite reagent (ATPLite PerkinElmer #6016731) was added. The plate was incubated in the dark at room temperature for 10 minutes. Luminescence was read spectrophotometrically using a VictorNivo (PerkinElmer) and data was expressed as a percentage of cell viability, with untreated control cells taken as 100%.

[0267] Example 12C. Luciferase assay. After 24 hours of incubation, 100 μl of BrightGlo reagent (Promega #E2620) was added into each well according to the manufacturer's instructions. After 5 minutes of incubation at room temperature, luciferase activity was measured using VictorNivo (Perkin Elmer). Data was expressed as luminescence versus percentage of transfection relative to the transfection positive control.

[0268] Example 12D. Biological activity of polyplexes formed by N1 and V1 in HeLa cells The transfection efficiency and cell viability of polyplexes formed by N1 and V1 in HeLa cells are reported in the following table. Transfection data are expressed as % of the positive control jetPEI®, which is 100% after 24 hours of treatment, and cell viability is compared to untreated (NT) cells, with the readout of the ATP content of NT cells being equal to 100%. [Table 31]

[0269] Example 12E. Biological activity of polyplexes formed by N1 and V2 in HeLa cells The transfection efficiency and cell viability of polyplexes formed by N1 and V2 in HeLa cells are reported in the following table. Transfection data are expressed as % of the positive control jetPEI®, which is 100% after 24 hours of treatment, and cell viability is compared to untreated (NT) cells, with the readout of the ATP content of NT cells being equal to 100%. [Table 32]

[0270] Example 12F. Biological activity of polyplexes formed by N1 and W1 in HeLa cells The transfection efficiency and cell viability of polyplexes formed by N1 and W1 in HeLa cells are reported in the following table. Transfection data are expressed as % of the positive control jetPEI®, which is 100% after 24 hours of treatment, and cell viability is compared to untreated (NT) cells, with the readout of ATP content in NT cells being equal to 100%. [Table 33]

[0271] Example 12G. Biological activity of polyplexes formed by N1 and V1 in HEK293 cells The transfection efficiency and cell viability of polyplexes formed by N1 and V1 in HEK293 cells are reported in the following table. Transfection data are expressed as % of the positive control jetPEI®, which is 100% after 24 hours of treatment, and cell viability is compared to untreated (NT) cells, with the readout of ATP content in NT cells being equal to 100%. [Table 34]

[0272] Example 12H. Biological activity of polyplexes formed by N1 and W1 in HEK293 cells The transfection efficiency and cell viability of polyplexes formed by N1 and W1 in HEK293 cells are reported in the following table. Transfection data are expressed as % of the positive control jetPEI®, which is 100% after 24 hours of treatment, and cell viability is compared to untreated (NT) cells, with the readout of ATP content in NT cells being equal to 100%. [Table 35]

[0273] As can be derived from the above data, the presence of the shielding polymer not only enhances cell viability and reduces the toxicity of the polymer complex in HEK293 cells, but also significantly increases the transfection efficiency by up to 6-fold. References J Mol Med, 2002, vol. 80, pp. 648-654 - TWGreen and PGM Wuts, Protective Groups in Organic Chemistry, Wiley, 3rd ed. 1999, Chapter 5 (pp. 369-451) - TWGreen and PGM Wuts, Protective Groups in Organic Chemistry, Wiley, 3rd ed. 1999, Chapter 7 (pp. 495-653) - Chinese Journal of Polymer Science (English Edition), 33(8), 1186-1195

[0274] Terms For reasons of completeness, the various aspects of the invention are set out in the following numbered clauses: Clause 1. Any stereoisomer or mixture of stereoisomers of any of the compounds of formula (Ia) or (Ib) or pharma- ceutically acceptable salts thereof, including anionic polymers of formula (Ia) or (Ib), or pharma- ceutically acceptable salts thereof, or homopolypeptides or random or block or graft copolypeptides; [ka] wherein Y is selected from the group consisting of -CO(CH2)p-CO- and -CO-(CH2)qSS-(CH2)r-CO-; Z is selected from the group consisting of a single bond, -CO-(CH2)qSS-(CH2)r-NH-(R5)z-, -CO-(CH2)p-NH-(R5)z-, and -(R5)z-; ○ is an integer selected from 1 to 2; In the formula, p, q, and r each independently represent an integer selected from 1 to 6; R5 is a random or block copolymer comprising at least two different repeat units selected from the group consisting of (II), (III), (IV), (V), (VI), (VII), (VIII), and (IX): [ka] a, b, c, d, e, f, g, and h are each independently an integer selected from 0 to 20; provided that at least one of a, b, c, d, e, f, g, and h is different from 0; z is an integer selected from 5 to 100; X is selected from N, S, and O; R1 is selected from the group consisting of H and (C1-C6)alkyl, with the proviso that R1 is absent when X is O; R3 and R4 are each independently selected from the group consisting of H and -CH3; m is an integer selected from 5 to 250; n is an integer selected from 3 to 200; However, the m:n ratio ranges from 1:8 to 30:1; R2 is selected from H and a radical selected from the group consisting of (X), (XI), (XII), and (XIII); [ka] In the formula, s, t, u, and v are each independently an integer selected from 1 to 4; wherein " / " indicates that the sequential order of the monomeric repeat units defined in the brackets on either side of the symbol is arbitrary, however, although the repeat units defined in the brackets in formulas Ia and Ib are shown in a particular order for convenience of illustration, the repeat units may be present in any order, and the repeat units may be present in blocks or randomly; wherein in formula Ib, the sequential order of the repeat units of each of formulae (II), (III), (IV), (V), (VI), (VII), (VIII), and (IX) in R5 and the repeat units indicated by the square brackets with the integer n may be present in blocks or randomly; A and A' are each H, OH, linear or branched -(C1-C6) alkyl, linear or branched -CO(C1-C6) alkyl, -(C5-C 10 )Aryl, -(C5-C 10 )Heteroaryl, -(C6-C 10 ) aralkyl, -(C1-C6) alkyl-O-(C5-C 10 )Aryl, -(C5-C 10 )heterocycloalkyl, -(C1-C 10 )Alkoxy, -(C6-C 10 )Aryloxy, -(C6-C 10 )Aralkoxy, -(C5~C 10 )Heteroaralkoxy, -(C1-C 10 )Alkyl-O-(C6-C 10 ) independently selected from aryloxy, an amine protecting group, a natural or unnatural alpha amino acid, a carboxyl protecting group, a labeling or imaging agent, and a cell targeting agent; each of A and A' is independently optionally substituted with one or more groups selected from the group consisting of -OH, halogen, -CF3, -NH2, -NH-(C1-C4)alkyl, NR6R7, -NH-CO-(C1-C6)alkyl, -(C1-C6)alkyl, -NO2, -N3, -CO-(C1-C6)alkyl, -CO-O-(C1-C6)alkyl, -SO3H, -SON2NH2, -SO2-N((C1-C6)alkyl)2, -COOH, CONH2, and -CON((C1-C6)alkyl)2; R6 and R7 are independently selected from the group consisting of H, -(C1-C4)alkyl, and an amine protecting group.

[0275] Clause 2. The anionic polymer according to clause 1, wherein m is an integer selected from 20 to 160 and n is an integer selected from 4 to 100, with the proviso that the ratio of m:n is in the range of 1:5 to 10:1.

[0276] Clause 3. The anionic polymer according to clause 1 or 2, which is an anionic polymer of formula (Ia) or (Ib), wherein R3 is -CH3 and R2 is H.

[0277] Clause 4. An anionic polymer of formula (Ib1), [ka] 4. The anionic polymer according to any one of clauses 1 to 3, wherein R2 is selected from the group consisting of radicals selected from the group consisting of (X), (XI), (XII), and (XIII).

[0278] Clause 5. An anionic polymer according to any one of clauses 1 to 3, which is an anionic polymer of formula (Ib1) as defined above, wherein R2 is H and R3 is -CH3.

[0279] Clause 6. The anionic polymer according to any one of clauses 1 to 5, which is of formula (Ib): wherein Z is selected from the group consisting of -CO-(CH2)qSS-(CH2)r-NH-(R5)z-, -CO-(CH2)p-NH-(R5)z-, and -(R5)z-; R5 is a random or block copolymer comprising at least two different repeat units selected from the group consisting of (II), (III), (IV), (V), (VI), (VII), (VIII), and (IX), wherein the sequential order of the repeat units of each of formulas (II), (III), (IV), (V), (VI), (VII), (VIII), and (IX) in R5 and the repeat units indicated in the square brackets with the integer n may be present in blocks or randomly.

[0280] Clause 7. The anionic polymer according to any of clauses 1-6, wherein the polymer is of formula Ib: wherein Z is selected from the group consisting of -CO-(CH2)qSS-(CH2)r-NH-(R5)z-, -CO-(CH2)p-NH-(R5)z-, and -(R5)z-; R5 is a random or block copolymer comprising at least two repeat units selected from the group consisting of (III), (VI), (VIII), and (IX): wherein c, e, g, and h are integers independently selected from 1 to 20; and z is an integer selected from 5 to 35.

[0281] Clause 8. The anionic polymer according to any one of clauses 1 to 7, which is of formula (Ib); wherein Z is selected from the group consisting of -CO-(CH2)qSS-(CH2)r-NH-(R5)z-, -CO-(CH2)p-NH-(R5)z-, and -(R5)z-; R5 is a random or block copolymer comprising at least two different repeat units selected from the group consisting of (III), (VI), (VIII), and (IX); c, e, g, and h are each independently an integer selected from 0 to 20; with the proviso that at least two of c, e, g, and h are different from 0; z is an integer selected from 5 to 35; wherein the sequential order of each repeat unit of R5 and the repeat units indicated by the square brackets with the integer n may be present in a block or random manner.

[0282] Clause 9. The anionic polymer according to any one of clauses 1 to 8, which is of formula (Ib): wherein Z is selected from the group consisting of -CO-(CH2)qSS-(CH2)r-NH-(R5)z-, -CO-(CH2)p-NH-(R5)z-, and -(R5)z-; R5 is a random or block copolymer comprising at least two different repeat units selected from the group consisting of (III), (VI), (VIII), and (IX); c, e, g, and h are are each independently an integer selected from 0 to 20; with the proviso that at least two of c, e, g, and h are different from 0; z is an integer selected from 5 to 35; m is an integer selected from 5 to 250, and n is an integer selected from 3 to 200, wherein the ratio of m:n ranges from 1:8 to 30:1, and wherein the sequential order of each repeat unit of R5 and the repeat units indicated in the square brackets with the integer n may be present in either blocks or randomly.

[0283] Clause 10. An anionic polymer according to any one of clauses 1 to 9 according to formula (Ib2), which is of formula (Ib) where Z is (R5)z: [ka] wherein b, e, f, g, and h are each independently an integer selected from 0 to 20; provided that at least one of b, e, f, g, and h is different from 0; z is an integer selected from 5 to 100; m is an integer selected from 5 to 250; n is an integer selected from 3 to 200; However, the m:n ratio ranges from 1:8 to 30:1; where the " / " between bracketed repeat units with integers b, e, n, f, g, and h indicates that the sequential order of the bracketed monomer repeat units on either side of the symbol is arbitrary; Here, the " / " between the bracketed repeat unit with the integer m and the bracketed repeat unit with the integer z+n indicates that the sequential order of the monomer repeat units on either side of the symbol is arbitrary.

[0284] Clause 11. An anionic polymer according to any one of clauses 1 to 3, which is of formula (Ia); R2 is selected from H and a radical selected from the group consisting of (X) and (XII), and R3 is -CH3.

[0285] Clause 12. The anionic polymer according to clause 7, wherein Y is -CO-(CH2)p-CO-; R2 is selected from the group consisting of H, (X) and (XII); R3 and R4 are H; m is an integer selected from 20 to 160; n is an integer selected from 4 to 100; and the ratio of m:n is in the range of 1:5 to 10:1.

Claims

1. Any stereoisomer or mixture of stereoisomers of a compound of formula (Ia) or (Ib), or of a pharmaceutically acceptable salt thereof, comprising an anionic polymer of formula (Ia) or (Ib), a pharmaceutically acceptable salt thereof, or a homopolypeptide or a random or block or graft copolymer of polypeptides 【Chemical 1】 (wherein Y is selected from the group consisting of -CO(CH ２ )p-CO- and -CO-(CH ２ )q-S-S-(CH ２ )r-CO-; Z is selected from the group consisting of -CO-(CH ２ )q-S-S-(CH ２ )r-NH-(R ５ )z-, -CO(CH ２ )pNH-(R ５ )z-, -CO-(CH ２ )p-CO-NH-(CH ２ )q-S-S-(CH ２ )r-NH-(R ５ )z-, and -(R ５ )z-; o is an integer selected from 1 to 2; wherein p, q, and r are each independently an integer selected from 1 to 6; R ５ is a random or block copolymer comprising at least two different repeating units selected from the group consisting of (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX): 【Chemical Formula 2】 a, b, c, d, e, f, g and h are each independently an integer selected from 0 to 20; provided that at least two of a, b, c, d, e, f, g and h are different from 0; z is an integer selected from 5 to 100; X is selected from N, S, and O; R １ is selected from the group consisting of H and (C １ ~C ６ ) alkyl, provided that R １ does not exist when X is O; R ３ and R ４ are each independently selected from the group consisting of H and -CH ３ ; m is an integer selected from 5 to 250; n is an integer selected from 3 to 200; provided that the ratio of m:n is in the range of 1:8 to 30:1; R ２ is selected from H and a radical selected from the group consisting of (X), (XI), (XII), and (XIII), 【Chemical Formula 3】 wherein s, t, u, and v are each independently an integer selected from 1 to 4; wherein " / " indicates that the consecutive order of the monomer repeating units defined by the square brackets on both sides of the symbol is arbitrary, but the repeating units defined by the square brackets in formulas Ia and Ib are shown in a specific order for the sake of explanation, and the repeating units may exist in any order and the repeating units may exist in blocks or randomly; In formula Ib, R ５ The respective repeating units of formulas (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX), and the consecutive order of the repeating units indicated by angle brackets with the integer n may be present in blocks or randomly; A and A' are each independently selected from H, OH, linear or branched -(C １ -C ６ ), alkyl, linear or branched -CO(C １ -C ６ ), alkyl, -(C ５ -C １０ ), aryl, -(C ５ -C １０ ), heteroaryl, -(C ６ -C １０ ), aralkyl, -(C １ -C ６ ), alkyl -O-(C ５ -C １０ ), aryl, -(C ５ -C １０ ), heterocycloalkyl, -(C １ -C １０ ), alkoxy, -(C ６ -C １０ ), aryloxy, -(C ６ -C １０ ), aralkoxy, -(C ５ -C １０ ), heteroaralkoxy, -(C １ -C １０ ), alkyl -O-(C ６ -C １０ ), aryloxy, an amine protecting group, a natural or non-natural alpha amino acid, a carboxyl protecting group, a labeling agent or imaging agent and a cell targeting agent; Each of A and A' is optionally substituted independently by one or more groups selected from the group consisting of -OH, halogen, -CF ３ , -NH ２ , -NH-(C １ ~C ４ )alkyl, NR ６ R ７ , -NH-CO-(C １ ~C ６ )alkyl, -(C １ ~C ６ )alkyl, -NO ２ , -N ３ , -CO-(C １ ~C ６ )alkyl, -CO-O-(C １ ~C ６ )alkyl, -SO ３ H, -SO ２ NH ２ , -SO ２ -N((C １ ~C ６ )alkyl) ２ , -COOH, CONH ２ , and -CON((C １ ~C ６ )alkyl) ２ ; and is optionally substituted independently R ６ and R ７ are each independently selected from the group consisting of H, -(C １ ~C ４ )alkyl, and amine protecting groups).

2. The anionic polymer according to claim 1, wherein m is an integer selected from 20 to 160 and n is an integer selected from 4 to 100; provided that the ratio of m:n is in the range of 1:5 to 10:

1.

3. Those of formula (Ia) or (Ib), wherein R ３ is -CH ３ and R ２ is H, the anionic polymer according to claim 1.

4. Of the formula (Ib), wherein Z is a single bond; R ２ is selected from H and a radical selected from the group consisting of (X), (XI), (XII), and (XIII); and R ３ is -CH ３ The anionic polymer according to claim 1, wherein is

5. of formula Ib; wherein Z is -CO-(CH ２ )q-S-S-(CH ２ )r-NH-(R ５ )z-, -CO(CH ２ )pNH-(R ５ )z-, -CO-(CH ２ )p-CO-NH-(CH ２ )q-S-S-(CH ２ )r-NH-(R ５ )z- and -(R ５ )z- selected from the group consisting of; and R ５ is a random or block copolymer comprising at least two repeating units selected from the group consisting of (III), (VI), (VIII) and (IX); and wherein c, e, g, and h are integers independently selected from 1 to 20; and z is an integer selected from 5 to 35, the anionic polymer according to claim 1.

6. It is of the formula (Ia); R ２ is selected from H and radicals selected from the group consisting of (X) and (XII); and R ３ is -CH ３ The anionic polymer according to claim 1, wherein it is

7. Y is -CO-(CH ２ )p-CO; R ２ is selected from the group consisting of H, (X), and (XII); R ３ and R ４ are H; m is an integer selected from 20 to 160; n is an integer selected from 4 to 100; provided that the ratio of m:n is in the range of 1:5 to 10:1, the anionic polymer according to claim 6.

8. a) A cationic polymer that binds covalently or electrostatically to at least one active agent selected from the group consisting of a pharmaceutically active agent, a veterinary active agent, a cosmetic active agent, a diagnostic active agent, a nucleic acid, a peptide, an antibody, an aptamer, a protein, and mixtures thereof, wherein the cationic polymer and the at least one active agent form positively charged nanoparticles; b) any stereoisomer or mixture of stereoisomers of a compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, comprising an anionic polymer of formula (Ia) or (Ib), a pharmaceutically acceptable salt thereof, or a homopolypeptide or a random or block or graft copolymer polypeptide that interacts electrostatically with the nanoparticles 【Chemical 4】 (wherein Y is selected from the group consisting of -CO(CH ２ )p-CO- and -CO-(CH ２ )q-S-S-(CH ２ )r-CO-; Z is selected from the group consisting of a single bond, -CO-(CH ２ ), q-S-S-(CH ２ ), r-NH-(R ５ ), z-, -CO(CH ２ ), pNH-(R ５ ), z-, -CO-(CH ２ ), p-CO-NH-(CH ２ ), q-S-S-(CH ２ ), r-NH-(R ５ ), z-, and -(R ５ ), z-; o is an integer selected from 1 to 2; wherein p, q, and r are each independently an integer selected from 1 to 6; R ５ is a random or block copolymer comprising at least two different repeating units selected from the group consisting of (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX): [Chemical Formula 5] a, b, c, d, e, f, g and h are each independently an integer selected from 0 to 20; provided that at least two of a, b, c, d, e, f, g and h are different from 0; z is an integer selected from 5 to 100; X is selected from N, S, and O; R １ is selected from the group consisting of H and (C １ ~C ６ ) alkyl, provided that R １ does not exist when X is O; R ３ and R ４ are each independently selected from the group consisting of H and -CH ３ ; m is an integer selected from 5 to 250; n is an integer selected from 3 to 200; provided that the ratio of m:n is in the range of 1:8 to 30:1; R ２ is selected from H and a radical selected from the group consisting of (X), (XI), (XII), and (XIII), 【Chemical Formula 6】 wherein s, t, u, and v are each independently an integer selected from 1 to 4; wherein " / " indicates that the consecutive order of the monomer repeating units defined by the square brackets on both sides of the symbol is arbitrary, but the repeating units defined by the square brackets in formulas Ia and Ib are shown in a specific order for convenience of explanation, and the repeating units may exist in any order and the repeating units may exist in blocks or randomly; in formula Ib, the consecutive order of each repeating unit of formula (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX) of R5 and the repeating unit shown in square brackets with the integer n may exist in blocks or randomly; A and A' are each independently selected from H, OH, linear or branched -(C １ ~C ６ ), alkyl, linear or branched -CO(C １ ~C ６ ), -(C ５ ~C １０ ), aryl, -(C ５ ~C １０ ), heteroaryl, -(C ６ ~C １０ ), aralkyl, -(C １ ~C ６ ), alkyl-O-(C ５ ~C １０ ), aryloxy, -(C ５ ~C １０ ), heterocycloalkyl, -(C １ ~C １０ ), alkoxy, -(C ６ ~C １０ ), aryloxy, -(C ６ ~C １０ ), aralkoxy, -(C ５ ~C １０ ), heteroaralkoxy, -(C １ ~C １０ ), alkyl-O-(C ６ ~C １０ ), aryloxy, an amine protecting group, a natural or non-natural alpha amino acid, a carboxyl protecting group, a labeling agent or imaging agent and a cell targeting agent; Each of A and A' is optionally independently substituted by one or more groups selected from the group consisting of -OH, halogen, -CF ３ , -NH ２ , -NH-(C １ ~C ４ )alkyl, NR ６ R ７ , -NH-CO-(C １ ~C ６ )alkyl, -(C １ ~C ６ )alkyl, -NO ２ , -N ３ , -CO-(C １ ~C ６ )alkyl, -CO-O-(C １ ~C ６ )alkyl, -SO ３ H, -SO ２ NH ２ , -SO ２ -N((C １ ~C ６ )alkyl) ２ , -COOH, CONH ２ , and -CON((C １ ~C ６ )alkyl) ２ ; R ６ and R ７ are each independently selected from the group consisting of H, —(C １ ~C ４ ) alkyl, and amine protecting groups) A polymer complex comprising.

9. The polymer complex according to claim 8, wherein the at least one active agent is selected from the group consisting of low molecular weight drugs, peptides, proteins, antibodies, nucleic acids, aptamers, and combinations thereof.

10. The polymer complex according to claim 9, wherein the nucleic acid is selected from the group consisting of DNA / RNA hybrids, short interfering RNA (siRNA), microRNA (miRNA), sgRNA, donor DNA, self-amplifying / replicating RNA, circular RNA (circRNA), plasmid DNA (pDNA), closed linear DNA (clDNA), short hairpin RNA (shRNA), messenger RNA (mRNA), and antisense RNA (aRNA), messenger RNA (mRNA), CRISPR guide RNA, antisense nucleic acid, decoy nucleic acid, aptamer, and ribozyme.

11. The polymer complex according to claim 10, wherein the nucleic acid is clDNA.

12. a) a cationically charged protein and b) at least one anionic polymer of formula (Ia) or (Ib), a pharmaceutically acceptable salt thereof, or a homopolypeptide or a random or block or graft copolymer polypeptide that electrostatically interacts with the protein, of any stereoisomer or mixture of stereoisomers of a compound of formula (Ia) or (Ib), or of a pharmaceutically acceptable salt thereof [Chemical Formula 7] (wherein Y is selected from the group consisting of -CO(CH ２ )p-CO- and -CO-(CH ２ )q-S-S-(CH ２ )r-CO-; Z is selected from the group consisting of a single bond, -CO-(CH ２ )q-SS-(CH ２ )r-NH-(R ５ )z-, -CO(CH ２ )pNH-(R ５ )z-, -CO-(CH ２ )p-CO-NH-(CH ２ )q-SS-(CH ２ )r-NH-(R ５ )z-, and -(R ５ )z-; o is an integer selected from 1 to 2; wherein p, q, and r are each independently an integer selected from 1 to 6; R ５ is a random or block copolymer comprising at least two different repeating units selected from the group consisting of (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX): 【Chemical Formula 8】 a, b, c, d, e, f, g, and h are each independently an integer selected from 0 to 20; provided that at least two of a, b, c, d, e, f, g, and h are different from 0; z is an integer selected from 5 to 100; X is selected from N, S, and O; R １ is selected from the group consisting of H and (C １ ~C ６ ) alkyl, provided that R １ does not exist when X is O; R ３ and R ４ are each independently selected from the group consisting of H and -CH ３ ; m is an integer selected from 5 to 250; n is an integer selected from 3 to 200; provided that the ratio of m:n is in the range of 1:8 to 30:1; R ２ is selected from H and a radical selected from the group consisting of (X), (XI), (XII), and (XIII), 【Chemical Formula 9】 wherein s, t, u, and v are each independently an integer selected from 1 to 4; wherein " / " indicates that the consecutive order of the monomer repeating units defined by the square brackets on both sides of the symbol is arbitrary, but the repeating units defined by the square brackets in formulas Ia and Ib are shown in a specific order for convenience of explanation, and the repeating units may exist in any order and the repeating units may exist in blocks or randomly; In formula Ib, the repeating units of each of formulas (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX) of R5 and the consecutive order of the repeating units shown in angle brackets with the integer n may exist in blocks or randomly; A and A' are each independently selected from H, OH, linear or branched -(C １ ~C ６ )-alkyl, linear or branched -CO(C １ ~C ６ )-alkyl, -(C ５ ~C １０ )-aryl, -(C ５ ~C １０ )-heteroaryl, -(C ６ ~C １０ )-aralkyl, -(C １ ~C ６ )-alkyl-O-(C ５ ~C １０ )-aryl, -(C ５ ~C １０ )-heterocycloalkyl, -(C １ ~C １０ )-alkoxy, -(C ６ ~C １０ )-aryloxy, -(C ６ ~C １０ )-aralkoxy, -(C ５ ~C １０ )-heteroaralkoxy, -(C １ ~C １０ )-alkyl-O-(C ６ ~C １０ )-aryloxy, an amine protecting group, a natural or non-natural alpha amino acid, a carboxyl protecting group, a labeling agent or imaging agent and a cell targeting agent; Each of A and A' is optionally independently substituted by one or more groups selected from the group consisting of -OH, halogen, -CF ３ , -NH ２ , -NH-(C １ ~C ４ )alkyl, NR ６ R ７ , -NH-CO-(C １ ~C ６ )alkyl, -(C １ ~C ６ )alkyl, -NO ２ , -N ３ , -CO-(C １ ~C ６ )alkyl, -CO-O-(C １ ~C ６ )alkyl, -SO ３ H, -SO ２ NH ２ , -SO ２ -N((C １ ~C ６ )alkyl) ２ , -COOH, CONH ２ , and -CON((C １ ~C ６ )alkyl) ２ ; and is optionally independently substituted R ６ and R ７ are each independently selected from the group consisting of H, —(C １ ~C ４ )alkyl and amine protecting groups) A protein-based complex, or alternatively a') an anionically charged protein covalently or electrostatically bound to a cationic polymer forming positively charged nanoparticles, b') at least one anionic polymer of formula (Ia) or (Ib), a pharmaceutically acceptable salt thereof, or a homopolypeptide or a random or block or graft copolymer polypeptide that electrostatically interacts with the positively charged nanoparticles of a'), any stereoisomer or mixture of stereoisomers of a compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof 【Chemical 10】 (wherein Y is selected from the group consisting of -CO(CH ２ )p-CO- and -CO-(CH ２ )q-S-S-(CH ２ )r-CO-; Z is selected from the group consisting of a single bond, -CO-(CH ２ ), q-S-S-(CH ２ ), r-NH-(R ５ ), z-, -CO(CH ２ ), pNH-(R ５ ), z-, -CO-(CH ２ ), p-CO-NH-(CH ２ ), q-S-S-(CH ２ ), r-NH-(R ５ ), z-, and -(R ５ ), z-; o is an integer selected from 1 to 2; wherein p, q, and r are each independently an integer selected from 1 to 6; R ５ is a random or block copolymer comprising at least two different repeating units selected from the group consisting of (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX): 【Chemical 11】 a, b, c, d, e, f, g and h are each independently an integer selected from 0 to 20; provided that at least two of a, b, c, d, e, f, g and h are different from 0; z is an integer selected from 5 to 100; X is selected from N, S, and O; R １ is selected from the group consisting of H and (C １ ~C ６ ) alkyl, provided that R １ does not exist when X is O; R ３ and R ４ are each independently selected from the group consisting of H and -CH ３ ; m is an integer selected from 5 to 250; n is an integer selected from 3 to 200; provided that the ratio of m:n is in the range of 1:8 to 30:1; R ２ is selected from H and a radical selected from the group consisting of (X), (XI), (XII), and (XIII), 【Chemical Formula 12】 wherein s, t, u, and v are each independently an integer selected from 1 to 4; wherein " / " indicates that the consecutive order of the monomer repeating units defined by the angle brackets on both sides of the symbol is arbitrary, but the repeating units defined by the angle brackets in formulas Ia and Ib are shown in a specific order for convenience of explanation, but the repeating units may exist in any order and the repeating units may exist in blocks or randomly; In formula Ib, the repeating units of each of formulas (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX) of R5 and the consecutive order of the repeating units shown in angle brackets with the integer n may exist in blocks or randomly; A and A' are each independently selected from H, OH, linear or branched -(C １ ~C ６ ), alkyl, linear or branched -CO(C １ ~C ６ ), -(C ５ ~C １０ ), aryl, -(C ５ ~C １０ ), heteroaryl, -(C ６ ~C １０ ), aralkyl, -(C １ ~C ６ ), alkyl - O -(C ５ ~C １０ ), aryl, -(C ５ ~C １０ ), heterocycloalkyl, -(C １ ~C １０ ), alkoxy, -(C ６ ~C １０ ), aryloxy, -(C ６ ~C １０ ), aralkoxy, -(C ５ ~C １０ ), heteroaralkoxy, -(C １ ~C １０ ), alkyl - O -(C ６ ~C １０ ), aryloxy, an amine protecting group, a natural or non - natural alpha - amino acid, a carboxyl protecting group, a labeling agent or imaging agent, and a cell targeting agent; Each of A and A' is optionally substituted independently by one or more groups selected from the group consisting of -OH, halogen, -CF ３ , -NH ２ , -NH-(C １ ~C ４ )alkyl, NR ６ R ７ , -NH-CO-(C １ ~C ６ )alkyl, -(C １ ~C ６ )alkyl, -NO ２ , -N ３ , -CO-(C １ ~C ６ )alkyl, -CO-O-(C １ ~C ６ )alkyl, -SO ３ H, -SO ２ NH ２ , -SO ２ -N((C １ ~C ６ )alkyl) ２ , -COOH, CONH ２ , and -CON((C １ ~C ６ )alkyl) ２ ; and is optionally substituted independently R ６ and R ７ are each independently selected from the group consisting of H, -(C １ ~C ４ )alkyl, and amine protecting groups) A protein-based complex comprising [

13. ] A composition comprising at least one polymer complex according to claim 8 or at least one protein-based complex according to claim 12, together with one or more pharmaceutically, diagnostically, veterinarily or cosmetically acceptable excipients or carriers.

14. For use in a medicament a. The polymer complex according to claim 8, or alternatively b. The protein-based complex according to claim 12, or alternatively c. The composition according to claim 13 which is a therapeutic product.

15. A device for use in a method of delivering a nucleic acid or a protein into a cell, a tissue or an extracellular space, the device comprising a composition comprising the polymer complex according to claim 8, the protein-based complex according to claim 12, or the one according to claim 13.

16. Contacting a target cell, tissue or extracellular space with a solution comprising the polymer complex according to claim 8, the protein-based complex according to claim 12 or the pharmaceutical composition according to claim 13 such that the complex can be introduced into the target cell, tissue or extracellular space; moving the complex from an endosome into the cytoplasm; dissociating the complex in the cell; and releasing the nucleic acid or the protein into the cytoplasm; for use in a method of delivering a nucleic acid or a protein into a target cell, tissue or extracellular space, the polymer complex according to claim 8, the protein-based complex according to claim 12 or the pharmaceutical composition according to claim 13.

17. A method of transfecting a cell comprising contacting the cell with the polymer complex according to claim 8 and the protein-based complex according to claim 12.

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